Profiling of protease specificity using combinatorial fluorogenic substrate libraries

ABSTRACT

A method is presented for the preparation and use of fluorogenic peptide substrates that allows for the configuration of general substrate libraries to rapidly identify the primary and extended specificity of enzymes, such as proteases. The substrates contain a fluorogenic-leaving group, such as 7-amino-4-carbamoylmethyl-coumarin (ACC). Substrates incorporating the ACC leaving group show comparable kinetic profiles as those with the traditionally used 7-amino-4-methyl-coumarin (AMC) leaving group. The bifunctional nature of ACC allows for the efficient production of single substrates and substrate libraries using solid-phase synthesis techniques. The approximately 3-fold increased quantum yield of ACC over AMC permits reduction in enzyme and substrate concentrations. As a consequence, a greater number of substrates can be tolerated in a single assay, thus enabling an increase in the diversity space of the library. Soluble positional protease substrate libraries of 137,180 and 6,859 members, possessing amino acid diversity at the P4-P3-P2-P1 and P4-P3-P2 positions, respectively, were constructed. Employing this screening method the substrate specificities of a diverse array of proteases were profiled, including the serine proteases thrombin, plasmin, factor Xa, uPA, tPA, granzyme B, trypsin, chymotrypsin, human neutrophil elastase, and the cysteine proteases papain and cruzain. The resulting profiles create a pharmacophoric portrayal of the proteases allowing for the design of selective substrates and potent inhibitors.

CROSS REFERENCE TO RELATED APPLICATIONS

[0001] The present application claims priority to U.S. ProvisionalPatent Application Serial No. 60/209,274, filed on Jun. 2, 2000, thedisclosure of which is incorporated herein in its entirety for allpurposes.

STATEMENT AS TO RIGHTS TO INVENTIONS MADE UNDER FEDERALLY SPONSOREDRESEARCH AND DEVELOPMENT

[0002] This work was supported in part by National Institute of HealthGrants CA72006, AI35707, GM54051, and National Institute of HealthBiotechnology Grant Fellowship, and National Science Foundation GrantMCB9604379. The Government may have certain rights in this invention.

BACKGROUND OF THE INVENTION

[0003] The ability of an enzyme to discriminate among many potentialsubstrates is an important factor in maintaining the fidelity of mostbiological functions. While substrate selection can be regulated on manylevels in a biological context, such as spatial and temporallocalization of enzyme and substrate, concentrations of enzyme andsubstrate, and requirement of cofactors, the substrate specificity atthe enzyme active site is the overriding principle that determines theturnover of a substrate. Characterization of the substrate specificityof an enzyme clearly provides invaluable information for the dissectionof complex biological pathways. Definition of substrate specificity alsoprovides the basis for the design of selective substrates and inhibitorsto study enzyme activity.

[0004] Of the genomes that have been completely sequenced, 2% of thegene products encode proteases (Barrett, A. J., et al., (1998) Handbookof Proteolytic Enzymes (Academic Press, London)). This family of enzymesis crucial to every aspect of life and death of an organism. With theidentification of new proteases, there is a need for the development ofrapid and general methods to determine protease substrate specificity.While several biological methods, such as peptides displayed onfilamentous phage (Matthews, D. J., et al. (1993) Science 260:1113-7;Ding, L., et al., (1995) Proceedings of the National Academy of Sciencesof the United States of America 92:7627-31), and chemical methods, suchas support-bound combinatorial libraries (Lam, K. S., et al., (1998)Methods in Molecular Biology, 87:1-6), have been developed to identifyproteolytic substrate specificity, few offer the ability to rapidly andcontinuously monitor proteolytic activity against complex mixtures ofsubstrates in solution.

[0005] The use of 7-amino-4-methyl coumarin (AMC) fluorogenic peptidesubstrates is a well-established method for the determination ofprotease specificity (Zimmerman, M., et al., (1977) AnalyticalBiochemistry 78:47-51). Specific cleavage of the anilide bond liberatesthe fluorogenic AMC leaving group allowing for the simple determinationof cleavage rates for individual substrates. More recently, arrays (Lee,D., et al., (1999) Bioorganic and Medicinal Chemistry Letters 9:1667-72)and positional-scanning libraries (Rano, T. A., et al., (1997) Chemistryand Biology 4:149-55) of AMC peptide substrate libraries have beenemployed to rapidly profile the N-terminal specificity of proteases bysampling a wide range of substrates in a single experiment. Each ofthese published efforts was designed for profiling caspases, cysteineproteases that require an Asp residue at the P1-position for substrateturnover. This requirement allows for the convenient attachment of theP1-Asp to the solid-support through the carboxylic acid side-chain.Since most proteases do not require P1-Asp/Glu for activity, librariesgenerated by these methods have limited applicability. Naturally,fluorogenic substrates that contain P1-amino acids that do not possessadequate side-chain functionality for attachment to a solid support in astraightforward manner (Gly, Leu, Val, Ile, Ala, Pro, Phe) will not beamenable to similar synthetic strategies.

[0006] Recently Fmoc-based synthesis methods to displace support-boundpeptides with nucleophiles in a final cleavage step to produceC-terminal modified peptides have been developed (Backes et al., (1999)Journal of Organic Chemistry 64:2322-2330). The preparation offluorogenic peptide substrates with any residue at the P1-position ispossible by the preparation of AMC-amino acid derivatives, which arethen used as nucleophiles to produce the AMC-peptide substrates (Backeset al. (2000) Nature Biotechnology 18(2): 187-193).

[0007] Support bound fluorogenic materials are also known in the art.For example, Adamczyk et al., Bioorg. Med. Chem. Lett., 9:217-220(1999), have disclosed resin-supported fluorophores prepared from a newN-hydroxysuccinimidyl resin. The resin-bound active esters were used toprepare conjugates with haptens, such as estriol, thyroxine, phenytoin,etc. As the fluorophore is transferred from the resin to the freehapten, the resin-bound fluorophores of Adamczyk et al. do notconstitute an appropriate starting point for the solid-phase synthesisof a peptide, nor is the use of the resin-bound fluorophore forderivatization of pre-formed peptides disclosed.

[0008] While the art provides a selection of methods that are useful forlabeling materials with fluorophores, a method for the solid-phasesynthesis of fluorogenic peptides, which begins with a resin-boundfluorophore, and materials that allow the method to be practiced, wouldrepresent a significant advance in the art. Such a method has greatutility and provides a general strategy for the preparation offluorogenic peptide substrate libraries. An innovative method would meetthe following objectives: (1) the solid-phase synthesis method shouldenable direct incorporation of at least all 20 proteinogenic amino acidsat every position, including the P1-position; (2) the method should becompatible with art-recognized solid-phase peptide synthesis protocolsand instrumentation; and (3) the method should be flexible enough toenable the rapid synthesis of any single substrate, substrate array, andpositional scanning library. Quite surprisingly, the present inventionprovides such a method.

SUMMARY OF THE INVENTION

[0009] The present invention provides, for the first time, a highlyefficient method for the preparation of fluorogenic compound libraries,particularly peptide substrate libraries based upon a new bifunctionalfluorogenic-leaving group. The leaving group of the invention isexemplified by 7-amino-4-carbamoylmethyl-coumarin (ACC). In anillustrative embodiment, using Fmoc-synthesis protocols, all 20proteinogenic amino acids can be directly coupled to the support boundACC-leaving group to provide general sets of substrates for analyzingprotease substrate specificity. The versatility of the solid-phasesynthesis strategy allows for substrate-arrays (Lee, D., et al., (1999)Bioorganic and Medicinal Chemistry Letters 9:1667-72) and positionalscanning libraries (Rano, T. A., et al., (1997) Chemistry and Biology4:149-55) of any configuration to be rapidly prepared. The substratespecificity of numerous representative serine and cysteine proteaseswere profiled to show the utility and generality of libraries generatedby the ACC method.

[0010] Thus, in a first aspect, the present invention provides amaterial having the structure:

[0011] wherein: R¹, R², R³, R⁴, R⁵ and R⁶ are members independentlyselected from the group consisting of H, halogen, —NO₂, —CN,—C(O)_(m)R⁷, —C(O)NR⁸R⁹, —S(O)_(t)R¹⁰, —SO₂NR¹¹R¹², —OR¹³, substitutedor unsubstituted alkyl, —R¹⁴-SS, and —NHR¹⁵ with the proviso that atleast one of R¹, R², R³, R⁴, R⁵ and R⁶ is —R¹⁴-SS and at least one ofR¹, R², R³, R⁴, R⁵ and R⁶ is —NHR¹⁵. R⁷, R⁸, R⁹, R¹⁰, R¹¹, R¹² and R¹³are members independently selected from the group consisting of H,substituted or unsubstituted alkyl and substituted or unsubstitutedaryl. R¹⁴ is a linking group adjoining the fluorogenic moiety and thesolid support. R¹⁵ is a member selected from the group consisting ofamine protecting groups, —C(O)-AA and —C(O)-P. P is a peptide sequence.AA is an amino acid residue. The subscript m is a member selected fromthe group consisting of the integers 1 and 2. The subscript t is amember selected from the group consisting of the integers from 0 to 2;and SS is a solid support.

[0012] In a second aspect, the present invention provides a fluorogenicpeptide comprising a fluorogenic moiety covalently bound to a peptidesequence. The peptide includes the structure:

R-P  (VII)

[0013] wherein, P is a peptide sequence having a structure that issubstantially identical to that set forth in Formula II. R is afluorogenic moiety having a structure substantially similar to thefluorogenic moiety of Formula I. The fluorogenic group substituents, R¹,R², R³, R⁴, R⁵ and R⁶, are members independently selected from the groupconsisting of H, halogen, —NO₂, —CN, —C(O)_(m)R⁷,—C(O)NR⁸R⁹,—S(O)_(t)R¹⁰, —SO₂NR¹¹R¹², —OR¹³, substituted or unsubstituted alkyl,—NHC(O)-P, and —R²⁰—Y. At least one of R¹, R², R³, R⁴, R⁵ and R⁶ is—R²⁰—Y and at least one of R¹, R², R³, R⁴, R⁵ and R⁶ is —NHC(O)-P. R⁷,R⁸, R⁹, R¹⁰, R¹¹, R¹² and R¹³ are members independently selected fromthe group consisting of H, substituted or unsubstituted alkyl andsubstituted or unsubstituted aryl. R²⁰ is either present or absent, andwhen present, is a member selected from the group consisting ofsubstituted or unsubstituted alkyl and substituted or unsubstitutedheteroalkyl; when R²⁰ is absent, Y is attached directly to thefluorogenic moiety. Y is an organic functional group or methyl, and ispreferably a member selected from the group consisting of —COOR¹⁷R²¹,CONR¹⁷R²¹, —C(O)R¹⁷, —OR¹⁷, —SR¹⁷, —NR¹⁷R²¹, —C(O)NR¹⁷R²¹, and—C(O)SR¹⁷. R¹⁷ and R²¹ are members independently selected from H,substituted or unsubstituted alkyl and substituted or unsubstitutedaryl. The subscript m is a member selected from the group consisting ofthe integers 1 and 2; and t is a member selected from the groupconsisting of the integers from 0 to 2.

[0014] In a further aspect, the present invention provides a library offluorogenic peptides having a structure according to Formula VII. Thelibrary includes at least a first peptide having a first peptidesequence covalently attached to a first fluorogenic moiety and a secondpeptide having a second peptide sequence covalently attached to a secondfluorogenic moiety. For each of each of the peptides of the library, Pis independently selected from peptide sequences, preferably having thestructure:

—C(O)-AA¹-AA²-(AA^(i))_(J-2)  (II).

[0015] Each of AA¹ through AA^(i) is an amino acid residue which is amember independently selected from the group consisting of natural aminoacid residues, unnatural amino acid residues and modified amino acidresidues. Each J is independently selected and denotes the number ofamino acid residues forming the first peptide sequence and the secondpeptide sequence and is a member selected from the group consisting ofthe numbers from 1 to 10. J can have the same value for each of thepeptide sequences in a particular library, or it can have a differentvalue for two or more of the peptides of the library. Each i isindependently selected and denotes the position of the amino acidresidue relative to AA¹ and when J is greater than 2, i is a memberselected from the group consisting of the numbers from 3 to 10.

[0016] For each of the peptides of the library, R is independentlyselected from fluorogenic moieties having a structure according toFormula I. Thus, the fluorogenic group(s) can be the same for each ofthe peptides of a particular library or the structure of R can vary in aselected manner for two or more peptides of the library.

[0017] For each of the library peptides having a structure according toFormula I, the substituents of the fluorogenic group, R¹, R², R³, R⁴,R⁵, and R⁶ are independently selected from the group consisting of H,halogen, —NO₂, —CN, —C(O)_(m)R⁷, —C(O)NR⁸R⁹, —S(O)_(t)R¹⁰, —SO₂NR¹¹R¹²,—OR¹³, substituted or unsubstituted alkyl, —NH—C(O)-P, R²⁰—Y, and—R¹⁴-SS. For each library peptide, at least one of R¹, R², R³, R⁴, R⁵,and R⁶ is a member independently selected from —R¹⁴-SS and —R²⁰—Y and atleast one of R¹, R², R³, R⁴, R⁵, and R⁶ is —NH—C(O)-P. R⁷, R⁸, R⁹, R¹⁰,R¹¹, R¹² and R¹³ for each library peptide are members independentlyselected from the group consisting of H, substituted or unsubstitutedalkyl and substituted or unsubstituted aryl. R¹⁴ is a linking groupadjoining the fluorogenic moiety and the solid support. R²⁰ is eitherpresent or absent, and when present, is a member selected from the groupconsisting of substituted or unsubstituted alkyl, and substituted orunsubstituted heteroalkyl; when R²⁰ is absent, Y is attached directly tothe fluorogenic moiety. The subscript m is a member selected from thegroup consisting of the integers from 1 to 2. The subscript t is amember selected from the group consisting of the integers from 0 to 2. Yis an organic functional group or methyl, and is preferably a memberselected from the group consisting of —COOR¹⁷, CONR¹⁷R²¹, —C(O)R¹⁷,—OR¹⁷, —SR¹⁷, NR¹⁷R²¹, —C(O)NR¹⁷R²¹, and —C(O)SR¹⁷. For each librarypeptide, R¹⁷ and R²¹ are members independently selected from the groupconsisting of H and substituted or unsubstituted alkyl. SS is a solidsupport.

[0018] Other objects and advantages of the present invention will beapparent from the Detailed Description, which follows.

BRIEF DESCRIPTION OF THE DRAWINGS

[0019]FIG. 1 Synthesis of 7-amino-4-carbamoylmethyl-coumarin substrates.(SPPS represents Solid-Phase Peptide Synthesis using standard Fmocprotocols).

[0020]FIG. 2 ACC P1-Diverse Library. The library consists of 20 wellswith 6,859 compounds per well (137,180 compounds total). The Y-axis isthe pM of fluorophore released per second. The X-axis provides thespatial address of the amino acid as represented by the one letter code(with “n” representing norleucine). P1-profiles of several serine andcysteine proteases:

[0021] A. Chymotrypsin; B. Trypsin; C. Thrombin; D. Plasmin; E. GranzymeB; F. Human Neutrophil Elastase; G. Papain; and H. Cruzain.

[0022]FIG. 3 Profiles of serine and cysteine proteases against P1-fixedACC PS-SCL. The Y-axis is the pM of fluorophore released per second. TheX-axis provides the spatial address of the amino acid as represented bythe one letter code (with “n” representing norleucine).

[0023] A. Lys, Plasmin; B. Arg, Thrombin; C. Arg, uPA; D. Arg, tPA; E.Arg, Factor Xa; F. Arg, Papain; G. Arg, Cruzain; H. Leu, Cruzain.

[0024]FIG. 4 Coomassie-stained gel of βI and βII tryptase expressionproducts. A. Recombinant βI tryptase, non-, single-, double-, andhyper-glycosylation forms are observed. B. Recombinant βII tryptase,non-, and single-glycosylation forms are observed. C. Native β-tryptase.D. Molecular mass standards.

[0025]FIG. 5 Results from the P1-Diverse positional scanning librarywhere the y-axis represents the rate of substrate cleavage (fluorophorerelease) over time and the x-axis represents the P1-amino acid. The P2,P3 and P4 positions contain an equimolar mixture of 19 amino acids (Cysand Met excluded, Nle included) for a total of 6,859 substrates/well.

[0026]FIG. 6 Results from the P1-Lys (A) and the P1-Arg (B) librarieswhere the y-axis represents the rate of substrate cleavage (fluorophorerelease) over time and the x-axis represents the positioned P2-, P3- orP4-amino acid. The two positions in the substrate that are not heldconstant contain an equimolar mixture of 19 amino acids (Cys and Metexcluded, Nle included) for a total of 361 substrates/well.

[0027]FIG. 7 Structural model of Ac-Pro-Arg-Asn-Lys-Nme substrateinteraction with tryptase. Two protomers are shown in green and orange.Two docked substrates are shown in magenta and white. Solvent-accessiblesurface of enzyme shown in (A). Figures prepared using Sybyl.

DETAILED DESCRIPTION OF THE INVENTION AND THE PREFERRED EMBODIMENTS

[0028] Abbreviations and Definitions

[0029] All technical and scientific terms used herein generally have thesame meaning as commonly understood by one of ordinary skill in the artto which this invention belongs. The present definitions andabbreviations are generally offered to supplement the art-recognizedmeanings. Generally, the nomenclature used herein and the laboratoryprocedures organic chemistry, enzyme chemistry and peptide synthesisdescribed below are those well known and commonly employed in the art.Generally, enzymatic reactions and purification steps are performedaccording to the manufacturer's specifications. Standard techniques, ormodifications thereof, are used for chemical syntheses and chemicalanalyses.

[0030] “AMC,” as used herein refers to, 7-amino-4-methyl-coumarin.

[0031] “ACC,” as used herein refers to,7-amino-4-carbamoylmethyl-coumarin.

[0032] “RFU,” as used herein refers to, relative fluorescence units.

[0033] “n” and “Nle,” as used herein refer to, norleucine.

[0034] “PS-SCL,” as used herein refers to, positional scanning-syntheticcombinatorial library;

[0035] “MUGB,” as used herein refers to, 4-methylumbelliferylp-guanidinobenzoate.

[0036] “Tris,” as used herein refers to,tris-(hydroxymethyl)-amino-methane.

[0037] “DIC,” as used herein refers to, diisopropylcarbodiimide.

[0038] “HOBt,” as used herein refers to, 1-hydroxybenzotriazole.

[0039] “TFA,” as used herein refers to, trifluoroacetic acid.

[0040] “Fmoc,” as used herein refers to, 9-fluorenylmethoxycarbonyl.

[0041] “pbf,” as used herein refers to,2,2,4,6,7-pentamethyldihydrobenzofuran-5-sulfonyl.

[0042] “trt,” as used herein refers to, trityl.

[0043] “Boc,” as used herein refers to, tert butoxycarbonyl.

[0044] “DMF,” as used herein refers to, N,N-dimethylformamide.

[0045] “NMP,” as used herein refers to, N-methylpyrrolidine.

[0046] “TIS,” as used herein refers to, triisopropylsilane.

[0047] “pbf,” as used herein refers to,2,2,4,6,7-pentamethyldihydrobenzofuran-5-sulfonyl.

[0048] “trt,” as used herein refers to, trityl.

[0049] “HATU,” as used herein refers to,O-(7-azabenzotriazol-1-yl)-1,1,3,3-tetramethyluroniumhexafluorophosphate.

[0050] The term “alkyl,” by itself or as part of another substituent,means, unless otherwise stated, a straight or branched chain, or cyclichydrocarbon radical, or combination thereof, which may be fullysaturated, mono- or poly-unsaturated and can include di- andmulti-valent radicals, having the number of carbon atoms designated(i.e. C₁-C₁₀ means one to ten carbons). Examples of saturatedhydrocarbon radicals include groups such as methyl, ethyl, n-propyl,isopropyl, n-butyl, t-butyl, isobutyl, sec-butyl, cyclohexyl,(cyclohexyl)ethyl, cyclopropylmethyl, homologs and isomers of, forexample, n-pentyl, n-hexyl, n-heptyl, n-octyl, and the like. Anunsaturated alkyl group is one having one or more double bonds or triplebonds. Examples of unsaturated alkyl groups include vinyl, 2-propenyl,crotyl, 2-isopentenyl, 2-(butadienyl), 2,4-pentadienyl,3-(1,4-pentadienyl), ethynyl, 1- and 3-propynyl, 3-butynyl, and thehigher homologs and isomers. The term “alkyl,” unless otherwise noted,is also meant to include those derivatives of alkyl defined in moredetail below as “heteroalkyl,” “cycloalkyl” and “alkylene.” The term“alkylene” by itself or as part of another substituent means a divalentradical derived from an alkane, as exemplified by —CH₂CH₂CH₂CH₂—.Typically, an alkyl group will have from 1 to 24 carbon atoms, withthose groups having 10 or fewer carbon atoms being preferred in thepresent invention. A “lower alkyl” or “lower alkylene” is a shorterchain alkyl or alkylene group, generally having eight or fewer carbonatoms.

[0051] “Substituted alkyl” refers to alkyl as just described includingone or more substituents such as, for example, lower alkyl, aryl, acyl,halogen (i.e., alkylhalos, e.g., CF₃), hydroxy, amino, alkoxy,alkylamino, acylamino, thioamido, acyloxy, aryloxy, aryloxyalkyl,mercapto, thia, aza, oxo, both saturated and unsaturated cyclichydrocarbons, heterocycles and the like. These groups may be attached toany carbon or substituent of the alkyl moiety. Additionally, thesegroups may be pendent from, or integral to, the alkyl chain.

[0052] The term “heteroalkyl,” by itself or in combination with anotherterm, means, unless otherwise stated, a stable straight or branchedchain, or cyclic hydrocarbon radical, or combinations thereof,consisting of the stated number of carbon atoms and from one to threeheteroatoms selected from the group consisting of O, N, Si and S, andwherein the nitrogen and sulfur atoms may optionally be oxidized and thenitrogen heteroatom may optionally be quaternized. The heteroatom(s) O,N and S may be placed at any interior position of the heteroalkyl group.The heteroatom Si may be placed at any position of the heteroalkylgroup, including the position at which the alkyl group is attached tothe remainder of the molecule. Examples include —CH₂—CH₂—O—CH₃,—CH₂—CH₂—NH—CH₃, —CH₂—CH₂—N(CH₃)—CH₃, —CH₂—S—CH₂—CH₃, —CH₂—CH₂—S(O)—CH₃,—CH₂—CH₂—S(O)₂—CH₃, —CH═CH—O—CH₃, —Si(CH₃)₃, —CH₂—CH═N—OCH₃, and—CH═CH—N(CH₃)—CH₃. Up to two heteroatoms may be consecutive, such as,for example, —CH₂—NH—OCH₃ and —CH₂—O—Si(CH₃)₃. Also included in the term“heteroalkyl” are those radicals described in more detail below as“heteroalkylene” and “heterocycloalkyl.” The term “heteroalkylene” byitself or as part of another substituent means a divalent radicalderived from heteroalkyl, as exemplified by —CH₂—CH₂—S—CH₂CH₂— and—CH₂—S—H₂—CH₂—NH—CH₂—. For heteroalkylene groups, heteroatoms can alsooccupy either or both of the chain termini. Still further, for alkyleneand heteroalkylene linking groups, no orientation of the linking groupis implied.

[0053] The term “aryl” is used herein to refer to an aromaticsubstituent, which may be a single aromatic ring or multiple aromaticrings which are fused together, linked covalently, or linked to a commongroup such as a diazo, methylene or ethylene moiety. The common linkinggroup may also be a carbonyl as in benzophenone. The aromatic ring(s)may include phenyl, naphthyl, biphenyl, diphenylmethyl and benzophenoneamong others. The term “aryl” encompasses “arylalkyl” and “substitutedaryl.”

[0054] “Substituted aryl” refers to aryl as just described including oneor more functional groups such as lower alkyl, acyl, halogen, alkylhalos(e.g. CF₃), hydroxy, amino, alkoxy, alkylamino, acylamino, acyloxy,phenoxy, mercapto and both saturated and unsaturated cyclic hydrocarbonswhich are fused to the aromatic ring(s), linked covalently or linked toa common group such as a diazo, methylene or ethylene moiety. Thelinking group may also be a carbonyl such as in cyclohexyl phenylketone. The term “substituted aryl” encompasses “substituted arylalkyl.”

[0055] The term “acyl” is used to describe a ketone substituent, —C(O)R,where R is alkyl or substituted alkyl, aryl or substituted aryl asdefined herein.

[0056] The term “halogen” is used herein to refer to fluorine, bromine,chlorine and iodine atoms.

[0057] The term “hydroxy” is used herein to refer to the group —OH.

[0058] The term “amino” is used to —NRR′, wherein R and R′ areindependently H, alkyl, aryl or substituted analogues thereof. “Amino”encompasses “alkylamino” denoting secondary and tertiary amines and“acylamino” describing the group RC(O)NR′.

[0059] The term “alkoxy” is used herein to refer to the —OR group, whereR is alkyl, or a substituted analogue thereof. Suitable alkoxy radicalsinclude, for example, methoxy, ethoxy, t-butoxy, etc.

[0060] As used herein, the term “linking group” refers to a group thatlinks a fluorogenic moiety to a solid support. Linking groups of diversestructures are useful in practicing the present invention. Exemplarylinking groups include, but are not limited to, organic functionalgroups (e.g., —C(O)—, —NR—, —C(O)S—, —C(O)NR—, etc.); substituted orunsubstituted alkyl, substituted or unsubstituted heteroalkyl andsubstituted or unsubstituted aryl groups each of which are, in additionto other optional substituents, homo- or hetero-disubstituted withorganic functional groups, that adjoin the linker arm to the fluorophoreand to the solid support. The linking groups of the invention caninclude a group that is cleaved by, for example, light, heat, reduction,oxidation, hydrolysis or enzymatic action (e.g., nitrophenyl, disulfide,ester, etc.). Alternatively, the linking group is substantially stableunder a range of conditions. By providing for the use of linkers with awide range of physicochemical characteristic, the invention allowsselected properties of the material of the invention and its conjugatesto be manipulated. Properties that are amenable to manipulation include,for example, hydrophobicity, hydrophilicity, surface-activity and thedistance from the solid support of the species bound to the solidsupport via the linking group.

[0061] “Peptide” refers to a polymer in which the monomers are aminoacids and are joined together through amide bonds, alternativelyreferred to as a polypeptide. When the amino acids are a-amino acids,either the L-optical isomer or the D-optical isomer can be used.Additionally, unnatural amino acids, for example, β-alanine,phenylglycine and homoarginine are also included. Commonly encounteredamino acids that are not gene-encoded may also be used in the presentinvention. All of the amino acids used in the present invention may beeither the D- or L-isomer. The L-isomers are generally preferred. Inaddition, other peptidomimetics are also useful in the presentinvention. For a general review, see, Spatola, A. F., in CHEMISTRY ANDBIOCHEMISTRY OF AMINO ACIDS, PEPTIDES AND PROTEINS, B. Weinstein, eds.,Marcel Dekker, New York, p. 267 (1983).

[0062] “Fluorogen,” as used herein, refers broadly to a class ofcompounds capable of being modified enzymatically or otherwise to give aderivative fluorophore, which has a modified or an increasedfluorescence.

[0063] “Solid support,” as used herein refers to a material that issubstantially insoluble in a selected solvent system, or which can bereadily separated (e.g., by precipitation) from a selected solventsystem in which it is soluble. Solid supports useful in practicing thepresent invention can include groups that are activated or capable ofactivation to allow selected species to be bound to the solid support. Asolid support can also be a substrate, for example, a chip, wafer orwell, onto which an individual, or more than one compound, of theinvention is bound.

[0064] “Organic functional group,” as used herein refers to groupsincluding, but not limited to, olefins, acetylenes, alcohols, phenols,ethers, oxides, halides, aldehydes, ketones, carboxylic acids, esters,amides, cyanates, isocyanates, thiocyanates, isothiocyanates, amines,hydrazines, hydrazones, hydrazides, diazo, diazonium, nitro, nitriles,mercaptans, sulfides, disulfides, sulfoxides, sulfones, sulfonic acids,sulfinic acids, acetals, ketals, anhydrides, sulfates, sulfenic acidsisonitriles, amidines, imides, imidates, nitrones, hydroxylamines,oximes, hydroxamic acids thiohydroxamic acids, allenes, ortho esters,sulfites, enamines, ynamines, ureas, pseudoureas, semicarbazides,carbodiimides, carbamates, imines, azides, azo compounds, azoxycompounds, and nitroso compounds. Methods to prepare each of thesefunctional groups are well-known in the art and their application to ormodification for a particular purpose is within the ability of one ofskill in the art (see, for example, Sandler and Karo, eds. ORGANICFUNCTIONAL GROUP PREPARATIONS, Academic Press, San Diego, 1989).

[0065] Introduction

[0066] The present invention provides a new fluorogenic leaving groupthat is attached to a solid support (e.g., acid-labile Rink linker) toprovide a solid support useful for solid-phase synthesis of diversemonomeric, oligomeric and polymeric materials. Also provided arecompounds to which the fluorogenic leaving group is attached librariesof such compounds and methods of using these compounds and libraries.

[0067] The invention alleviates many of the difficulties associated withart-recognized methods of forming fluorogenic compounds. For example,using the solid support of the invention, fluorogenic peptides havingsubstantially any amino acid residue at the carboxy-terminus (“P1”) areeasily prepared. The ability to prepare peptide libraries havingcomplete diversity at P1 using solid-phase techniques eliminates thewell known shortcomings of solution synthesis techniques, speeding bothsynthesis and purification.

[0068] Solid Supports

[0069] Synthesis on solid supports, “solid-phase synthesis,” is ofrecognized utility in the synthesis of small molecules, oligomericcompounds and polymers. A diverse array of solid supports bearing usefulprobes, labels and reactive groups are known in the art (see, forexample, Burgess, ed., SOLID-PHASE ORGANIC SYNTHESIS, John Wiley andSons, 2000; and Chan and White, eds., FMOC SOLID PHASE PEPTIDESYNTHESIS: A PRACTICAL APPROACH (The Practical Approach Series), OxfordUniversity Press, 2000. Solid supports include substantially anyoligomeric or polymeric material upon which a selected synthesis can beperformed, and the materials and methods of the present invention arenot limited by the identity of the material serving as the solidsupport.

[0070] Thus, in a first aspect, the present invention provides amaterial having the structure:

[0071] wherein: R¹, R², R³, R⁴, R⁵ and R⁶ are members independentlyselected from the group consisting of H, halogen, —NO₂, —CN,—C(O)_(m)R⁷, —C(O)NR⁸R⁹, —S(O)_(t)R¹⁰, —SO₂NR¹¹R¹², —OR¹³, substitutedor unsubstituted alkyl, —R¹⁴-SS, and —NHR¹⁵ with the proviso that atleast one of R¹, R², R³, R⁴, R⁵ and R⁶ is —R¹⁴-SS and at least one ofR¹, R², R³, R⁴, R⁵ and R⁶ is —NHR¹⁵. R⁷, R⁸, R⁹, R¹⁰, R¹¹, R¹² and R¹³are members independently selected from the group consisting of H,substituted or unsubstituted alkyl and substituted or unsubstitutedaryl. R¹⁴ is a linking group adjoining the fluorogenic moiety and thesolid support. R¹⁵ is a member selected from the group consisting ofamine protecting groups, —C(O)-AA and —C(O)-P. P is a peptide sequence.AA is an amino acid residue. The subscript m is a member selected fromthe group consisting of the integers 1 and 2. The subscript t is amember selected from the group consisting of the integers from 0 to 2;and SS is a solid support.

[0072] In a presently preferred embodiment, the linking group, R¹⁴, isan organic functional group adjoining the fluorogenic moiety and thesolid support. In another preferred embodiment, R¹⁴ is member selectedfrom the group consisting of substituted or unsubstituted alkyl,substituted or unsubstituted heteroalkyl and substituted orunsubstituted aryl groups, which are homo- or hetero-disubstituted withfunctional groups adjoining the linker to both the fluorogenic moietyand the solid support. Linkers useful for forming conjugates betweensolid supports and other species are well known in the art (see, forexample, James, Tetrahedron 55: 4855-4946 (1999)).

[0073] In a preferred embodiment, the invention provides a materialaccording to Formula I, in which P is a peptide sequence comprising thestructure:

—C(O)-AA¹-AA²-(AA^(i))_(J-2)  (II)

[0074] wherein, AA¹-AA²-(AA^(i))_(J-2) is a peptide sequence. Each ofAA¹ through AA^(i) is an amino acid residue which is a memberindependently selected from the group of natural amino acid residues,unnatural amino acid residues and modified amino acid residues. Thesubscript J denotes the number of amino acid residues forming thepeptide sequence and is a member selected from the group consisting ofthe numbers from 2 to 10, such that J-2 is the number of amino acidresidues in the peptide sequence exclusive of AA¹-AA². The superscript idenotes the position of an amino acid residue relevant to AA¹. When J isgreater than 2, i is a member selected from the group consisting of thenumbers from 3 to 10.

[0075] In another preferred embodiment, the invention provides amaterial according to Formula I, in which R¹⁵ has the structure:

—C(O)-AA  (III)

[0076] In Formula III, AA is an amino acid residue selected from thegroup consisting of natural amino acids, unnatural amino acids andmodified amino acids.

[0077] In a still further preferred embodiment, the invention provides amaterial according to Formula I, which has the structure:

[0078] in which, the substituents R¹, R³, R⁴, R⁵ and R⁶ havesubstantially the same identities as set forth herein above inconjunction with the materials according to Formula I. The compoundsaccording to this embodiment can comprise the single peptide sequencedisplayed in Formula IV, or one or more additional peptide sequences,which are the same as or different than the peptide sequence of FormulaIV. Moreover, the materials of the invention can comprise an amino acidas displayed in Formula III in addition to one or more peptidesequences.

[0079] In another preferred embodiment, the material of the inventionhas the structure:

[0080] wherein, the substituents R¹, R³, R⁴ and R⁶ are substantiallyidentical to those substituents set forth in conjunction with thematerial of the invention according to Formula IV. Z is a linkingselected from —O—, —NR¹⁶— and —S—. R¹⁶ is preferably a member selectedfrom H and substituted or unsubstituted alkyl. The subscript crepresents an integer, which is preferably selected from 0 to 6.

[0081] In another preferred embodiment, the invention provides amaterial having the structure:

[0082] in which the identities of the substituents R¹, R³, and R⁴ andthe identity of the linking group Z are substantially as describedhereinabove.

[0083] The fluorogenic materials of the invention are also of use assolid supports for the synthesis of individual compounds other thanpeptides and libraries consisting of an array of individual compoundsother than peptides. Exemplary compounds that can be synthesized usingthe solid support of the invention include, but are not limited to,small molecules and oligomers (e.g., nucleic acids, lipids, saccharides,etc.). Thus, the present invention provides libraries of fluorogeniccompounds other than peptides.

[0084] Fluorogenic Compounds

[0085] Fluorogenic compounds are of use as probes for an array ofapplications, including structural elucidation of materials, substratespecificity of enzymes, hybridization of nucleic acids, substratetransformation, digestion or degradation of biomolecules, such aspeptides, nucleic acids, saccharides and the like. As discussed above,the present invention provides a solid support, which allows for theconjugation of a fluorogenic moiety to compounds of different types,which are synthesized on the solid support of the invention.

[0086] Thus, in a second aspect, the present invention provides afluorogenic peptide comprising a fluorogenic moiety covalently bound toa peptide sequence. The peptide includes the structure:

R-P  (VII)

[0087] wherein, P is a peptide sequence having a structure that issubstantially identical to that set forth in Formula II. R is afluorogenic moiety having a structure substantially similar to thefluorogenic moiety of Formula I.

[0088] In the present aspect of the invention, the fluorogenic groupsubstituents, R¹, R², R³, R⁴, R⁵ and R⁶, are members independentlyselected from the group consisting of H, halogen, —NO₂, —CN,—C(O)_(m)R⁷,—C(O)NR⁸R⁹, —S(O)_(t)R¹⁰, —SO₂NR¹¹R¹², —OR¹³, substituted orunsubstituted alkyl, —NHC(O)-P, and —R²⁰—Y. At least one of R¹, R², R³,R⁴, R⁵ and R⁶ is —R²⁰—Y and at least one of R¹, R², R³, R⁴, R⁵ and R⁶ is—NHC(O)-P. R⁷, R⁸, R⁹, R¹⁰, R¹¹, R¹² and R¹³ are members independentlyselected from the group consisting of H, substituted or unsubstitutedalkyl and substituted or unsubstituted aryl. R²⁰ is either present orabsent, and when present, is a member selected from the group consistingof substituted or unsubstituted alkyl and substituted or unsubstitutedheteroalkyl; when R²⁰ is absent, Y is attached directly to thefluorogenic moiety. Y is an organic functional group or methyl, and ispreferably a member selected from the group consisting of —COOR¹⁷R²¹,CONR¹⁷R²¹, —C(O)R¹⁷, —OR¹⁷, —SR¹⁷, NR¹⁷R²¹, and —C(O)SR¹⁷. R¹⁷ and R²¹are members independently selected from H, substituted or unsubstitutedalkyl and substituted or unsubstituted aryl. The subscript m is a memberselected from the group consisting of the integers 1 and 2; and t is amember selected from the group consisting of the integers from 0 to 2.

[0089] In a further preferred embodiment, the present invention providesa fluorogenic peptide having a structure substantially identical to thatset forth in Formula IV. The identities of the fluorogenic groupsubstituents, R¹, R³, R⁴, R⁵ and R⁶, are substantially identical tothose set forth for the peptides of the invention according to FormulaVII.

[0090] In another preferred embodiment, the invention provides a peptidehaving the structure:

[0091] wherein, c is a member selected from the group consisting of theintegers from 0 to 6.

[0092] In yet a further preferred embodiment, the invention provides afluorogenic peptide having the structure:

[0093] in which Y is substantially as described above.

[0094] The fluorogenic peptides of the invention preferably have apeptide sequence that includes at least one peptide bond cleavable by anenzyme, preferably a protease. Cleaving the peptide bond preferablyreleases the fluorogenic moiety from the peptide sequence, therebyproducing a fluorescent moiety and a peptide moiety. The peptide bond,which undergoes enzymatic cleavage can be located at any site within thepeptide sequence, but is preferably located at a peptide bond formedbetween an amine of the fluorogenic moiety and a carboxylic acid moietyof the peptide carboxy terminus.

[0095] The present invention also provides the ability to introduce anadditional element of diversity in the positional scanning combinatoriallibraries through the preparation of a peptide library consisting of aplurality of wells (preselected amino acids, can be omitted or included)addressing a fixed P1 amino acid. In an illustrative embodiment having20 wells, a tetrapeptide is prepared in which the P2-P3-P4 positions inthe library consist of an equimolar mixture of 19 amino acids (cysteineis omitted and norleucine is substituted for methionine) for a total of6,858 substrates per well and 137,180 substrates per library. Thepresent invention provides a further advantage in that, if members ofthe library are sparingly soluble under a particular set of conditions,to avoid insolubility of the substrates as well as to maintaink_(cat)/K_(m) conditions, the concentration for each individualsubstrate per well can be decreased to approximately 0.01 μM. Theincreased fluorescence of the ACC fluorophore of the invention, relativeto the AMC fluorophore, provides for the use of lower concentrations ofsubstrate than in art-recognized methods.

[0096] Compound Libraries

[0097] The synthesis and screening of chemical libraries to identifycompounds having useful biological and material properties is now acommon practice. Illustrative of the many different types of librariesthat have been prepared are libraries including collections ofoligonucleotides, oligopeptides, and small or large molecular weightorganic or inorganic molecules. See, Moran et al., PCT Publication WO97/35198, published Sep. 25, 1997; Baindur et al., PCT Publication WO96/40732, published Dec. 19, 1996; Gallop et al., J. Med. Chem.37:1233-51 (1994).

[0098] Thus, in a further aspect, the present invention provides alibrary of fluorogenic compounds. In a presently preferred embodiment,there is provided a library of fluorogenic peptides having a structureaccording to Formula VII.

R-P  (VII)

[0099] The library includes at least a first peptide having a firstpeptide sequence covalently attached to a first fluorogenic moiety and asecond peptide having a second peptide sequence covalently attached to asecond fluorogenic moiety. For each of each of the peptides of thelibrary, P is independently selected from peptide sequences preferablyhaving the structure:

—C(O)-AA¹-AA²-(AA^(i))_(J-2)  (II).

[0100] Each of AA¹ through AA^(i) is an amino acid residue which is amember independently selected from the group consisting of natural aminoacid residues, unnatural amino acid residues and modified amino acidresidues. Each J is independently selected and denotes the number ofamino acid residues forming the first peptide sequence and the secondpeptide sequence and is a member selected from the group consisting ofthe numbers from 1 to 10. J can have the same value for each of thepeptide sequences in a particular library, or it can have a differentvalue for two or more of the peptides of the library. Each i isindependently selected and denotes the position of the amino acidresidue relative to AA¹ and when J is greater than 2, i is a memberselected from the group consisting of the numbers from 3 to 10.

[0101] For each of the peptides of the library, R is independentlyselected from fluorogenic moieties having a structure according toFormula I. Thus, the fluorogenic group(s) can be the same for each ofthe members of a particular library or the structure of R can vary in aselected manner for two or more members of the library.

[0102] For each of the library peptides having a structure according toFormula I, the substituents of the fluorogenic group, R¹, R², R³, R⁴,R⁵, and R⁶ are independently selected from the group consisting of H,halogen, —NO₂, —CN, —C(O)R⁷, —C(O)NR⁸R⁹, —S(O)_(t)R¹⁰, —SO₂NR¹¹R¹²,—OR¹³, substituted or unsubstituted alkyl, —NH—C(O)-P, R²⁰—Y, and—R¹⁴-SS. For each library peptide, at least one of R¹, R², R³, R⁴, R⁵,and R⁶ is a member independently selected from —R¹⁴-SS and —R²⁰—Y and atleast one of R¹, R², R³, R⁴, R⁵, and R⁶ is —NH—C(O)-P. R⁷, R⁸, R⁹,R¹⁰,R¹¹, R¹² and R¹³ for each library peptide are members independentlyselected from the group consisting of H, substituted or unsubstitutedalkyl and substituted or unsubstituted aryl. R¹⁴ is a linking groupadjoining the fluorogenic moiety and the solid support. R²⁰ is eitherpresent or absent, and when present, is a member selected from the groupconsisting of substituted or unsubstituted alkyl, and substituted orunsubstituted heteroalkyl; when R²⁰ is absent, Y is attached directly tothe fluorogenic moiety. The subscript m is a member selected from thegroup consisting of the integers from 1 to 2. The subscript t is amember selected from the group consisting of the integers from 0 to 2. Yis an organic functional group or methyl and is preferably a memberselected from the group consisting of —COOR¹⁷, CONR¹⁷R²¹, —C(O)R¹⁷,—OR¹⁷, —SR¹⁷, —C(O)SR¹⁷ and NR¹⁷R²¹. For each library peptide, R¹⁷ andR²¹ are members independently selected from the group consisting of Hand substituted or unsubstituted alkyl. SS is a solid support.

[0103] In other preferred embodiments, the invention provides a libraryof fluorogenic peptides wherein, each of the peptides of the library hasan independently selected structure according to Formula IV. In thisembodiment the substituents on the fluorogenic group, R¹, R³, R⁴, R⁵ andR⁶, are independently selected for each of the library peptides and theyare substantially similar to those set forth hereinabove in conjunctionwith the description of the library peptides that include a structureaccording to Formula I. For those library peptides having a structureaccording to Formula IV, the value of c is independently selected foreach of the library peptides and it is a member selected from the groupconsisting of the integers from 0 to 6.

[0104] In a further preferred embodiment, the invention provides alibrary of peptides having structures independently selected frompeptides according to Formula VIII, and more preferably Formula IX.

[0105] As discussed above, each of the peptide sequences and peptidelengths of the peptides of a particular library are independentlyselected. Thus, in a preferred embodiment, each of peptides of thelibrary is characterized by a peptide sequence that is different thanthe peptide sequence of each of the other peptides. The differenceresides in peptide sequence, peptide length or both. Thus, a preferredlibrary of the invention is one wherein, an amino acid residue selectedfrom at least one member of AA¹, AA² . . . AA^(i) of the first peptideis a different amino acid residue than an amino acid residue at acorresponding position relative to AA¹ of the second peptide.

[0106] The peptide libraries of the invention are broadly characterizedby the presence of peptides of diverse structure within the library. Inan exemplary embodiment, the diversity in the peptides of the library isprovided by peptide sequences that have different amino acid residues atAA¹. Those of skill in the art will appreciate that the focus of thepresent discussion on diversity at AA¹ is for clarity of illustrationand is not intended to exclude those peptide sequences having diversityat positions other than AA¹ or those peptide sequences having diversityat positions in addition to AA¹.

[0107] Thus, in a preferred embodiment, the library is characterized byhaving at least six peptides having different peptide sequences wherein,AA¹ is a different amino acid residue in each of the different peptidesequences. In another preferred embodiment, the library includes atleast twelve peptides, and more preferably twenty peptides havingdifferent peptide sequences, in which AA¹ is a different amino acidresidue in each of the different peptide sequences.

[0108] The amino acid residue at AA¹ can be any amino acid residueselected from the group consisting of natural amino acids, unnaturalamino acids and modified amino acids. In a preferred embodiment, AA¹ isa member selected from the group consisting of Lys, Arg, Leu andcombinations thereof.

[0109] The peptides of the library can have a peptide sequence ofsubstantially any useful length for a selected purpose. Presentlypreferred peptide sequences are those in which J is a member selectedfrom the numbers from 4 to 8.

[0110] Many processes have been devised for the synthesis of librariesof peptides and peptide analogs, which are applicable to practicing thepresent invention (see, for example, Gordon and Kerwin, COMBINATORIALCHEMISTRY AND MOLECULAR DIVERSITY IN DRUG DISCOVERY, Wiley-Liss, NewYork, 1998).

[0111] Libraries of peptides and certain types of peptide mimetics,called “peptoids”, have been assembled and screened for a desirablebiological activity by a range of methodologies (see, Gordon et al., J.Med Chem., 37: 1385-1401 (1994). For example, the method of Geysen,(Bioorg. Med. Chem. Letters, 3: 397-404 (1993); Proc. Natl. Acad Sci.USA, 81: 3998 (1984)) employs a modification of Merrifield peptidesynthesis, wherein the C-terminal amino acid residues of the peptides tobe synthesized are linked to solid-support particles shaped aspolyethylene pins; these pins are treated individually or collectivelyin sequence to introduce additional amino-acid residues forming thedesired peptides. The peptides are then screened for activity withoutremoving them from the pins. The solid support of the invention can besimilarly formed and used as a solid support for the synthesis ofpeptide libraries or other libraries.

[0112] Houghton, Proc. Natl. Acad. Sci. USA, 82: 5131 (1985); Eichler etal., Biochemistry, 32: 11035-11041 (1993); and U.S. Pat. No. 4,631,211)utilize individual polyethylene bags (“tea bags”) containing C-terminalamino acids bound to a solid support. These are mixed and coupled withthe requisite amino acids using solid phase synthesis techniques. Thepeptides produced are then recovered and tested individually.

[0113] Fodor et al., Science, 251: 767 (1991), describe light-directed,spatially addressable parallel-peptide synthesis on a silicon wafer togenerate large arrays of addressable peptides that can be directlytested for binding to biological targets. The solid support of theinvention can be utilized in a similar manner.

[0114] In another combinatorial approach, equally applicable to thepresent invention, Huebner et al. (U.S. Pat. No. 5,182,366) disclosesfunctionalized polystyrene beads divided into portions, each of which isacylated with a desired amino acid; the bead portions are mixedtogether, then divided into portions each of which is re-subjected toacylation with a second amino acid producing dipeptides. By using thissynthetic scheme, exponentially increasing numbers of peptides areproduced in uniform amounts, which are then separately screened for abiological activity of interest.

[0115] Presently preferred uses for the peptide libraries of theinvention include their use in probing the reactivity and substratespecificity of enzymes, and in particular proteases. Thus, preferredlibraries are those in which at least one peptide sequence of thelibrary is cleavable by a protease into a fluorescent moiety and thepeptide sequence, or a fragment of the peptide sequence.

[0116] The present invention provides techniques for preparing andprobing peptide libraries having a wide range of sizes. Thus, in apreferred embodiment, the library includes at least 10 peptides, whereineach of the peptide sequences is a different peptide sequence. Morepreferably, the library includes at least 100 peptides, wherein each ofthe peptide sequences is a different peptide sequence, more preferablyat least 1,000 peptides, still more preferably, at least 10,000peptides, more preferably, at least 100,000 peptides, and even stillmore preferably, at least 1,000,000 peptides.

[0117] In another preferred embodiment, the library of the invention isprovided with a means by which a library member (e.g., peptide sequence)can be resolved from the other library members. Many such means fordeconvoluting a library of compounds are known in the art, including,for example, the use of tags, positional libraries, and ordered arrays.Thus, in a preferred embodiment, the library of the invention has afirst member located at a first region of a substrate and a secondmember located at a second region of a substrate.

[0118] Libraries in a positional or an ordered array motif are presentlypreferred. Such libraries permit the identification of peptides, orother compounds, that are associated with zones of activity locatedduring screening the library. Specifically, the library can be orderedso that the position of the peptide on the array corresponds to theidentity of the peptide. Thus, once an assay has been carried out, andthe position on the array determined for an active peptide, the identityof that peptide can be easily ascertained.

[0119] In another preferred embodiment, the present invention provides alibrary in a microarray format comprising n compounds distributed over nregions of a substrate. Preferably, each of the n compounds is adifferent compound. In a still further preferred embodiment, the ncompounds are patterned on the substrate in a manner that allows theidentity of the compound at each of the n locations to be ascertained.The microarray is patterned from essentially any type of fluorogenicmolecule of the invention, including, but not limited to, small organicmolecules, peptides, nucleic acids, carbohydrates, antibodies, enzymes,and the like.

[0120] A variety of methods are currently available for making arrays ofbiological molecules, such as arrays of antibodies, nucleic acidmolecules, peptides or proteins. The following discussion utilizes a DNAmicroarray as an exemplary microarray. This use of DNA is intended to beillustrative and not limiting. One of skill in the art will appreciatethat the following discussion is substantially applicable to formingmicroarrays of other fluorogenic compounds of the invention as well.

[0121] One method for making ordered arrays of compounds on a porousmembrane is a “dot blot” approach. In this method, a vacuum manifoldtransfers a plurality, e.g., 96, aqueous samples of a compound from 3millimeter diameter wells to a porous membrane. A common variant of thisprocedure is a “slot-blot” method in which the wells havehighly-elongated oval shapes.

[0122] The compound is immobilized on the porous membrane by, forexample, baking the membrane or exposing it to UV radiation. This is amanual procedure practical for making one array at a time and usuallylimited to 96 samples per array.

[0123] A more efficient technique employed for making ordered arrays ofcompounds uses an array of pins dipped into the wells, e.g., the 96wells of a microtitre plate, for transferring an array of samples to asubstrate, such as a porous membrane. One array includes pins that aredesigned to spot a membrane in a staggered fashion, for creating anarray of 9216 spots in a 22×22 cm area. See, Lehrach, et al.,HYBRIDIZATION FINGERPRINTING IN GENOME MAPPING AND SEQUENCING, GENOMEANALYSIS, Vol. 1, Davies et al, Eds., Cold Springs Harbor Press, pp.39-81 (1990).

[0124] An alternate method of creating ordered arrays of compounds isdescribed by Pirrung et al. (U.S. Pat. No. 5,143,854, issued 1992), andalso by Fodor et al., (Science, 251: 767-773 (1991)) for preparingarrays of nucleic acid sequences. The method involves synthesizingdifferent compounds at different discrete regions of a substrate. Arelated method has been described by Southern et al. (Genomics, 13:1008-1017 (1992)).

[0125] Khrapko, et al., DNA Sequence, 1: 375-388 (1991) describes amethod of making a compound matrix by spotting DNA onto a thin layer ofpolyacrylamide. The spotting is done manually with a micropipette.

[0126] When the library is associated with a substrate, the substratecan also be patterned using techniques such as photolithography(Kleinfield et al., J. Neurosci. 8:4098-120 (1998)), photoetching,chemical etching and microcontact printing (Kumar et al., Langmuir10:1498-511 (1994)). Other techniques for forming patterns on asubstrate will be readily apparent to those of skill in the art.

[0127] The size and complexity of the pattern on the substrate islimited only by the resolution of the technique utilized and the purposefor which the pattern is intended. For example, using microcontactprinting, features as small as 200 nm are layered onto a substrate. See,Xia, Y.; Whitesides, G., J. Am. Chem. Soc. 117:3274-75 (1995).Similarly, using photolithography, patterns with features as small as 1μm have been produced. See, Hickman et al., J. Vac. Sci. Technol.12:607-16 (1994).

[0128] The pattern can be printed directly onto the substrate or,alternatively, a “lift off” technique can be utilized. In the lift offtechnique, a patterned resist is laid onto the substrate, a compound islaid down in those areas not covered by the resist and the resist issubsequently removed. Resists appropriate for use with the substrates ofthe present invention are known to those of skill in the art. See, forexample, Kleinfield et al., J Neurosci. 8:4098-120 (1998). Followingremoval of the photoresist, a second compound, having a structuredifferent from the first compound can be bonded to the substrate onthose areas initially covered by the resist. Using this technique,substrates with patterns having regions of different chemicalcharacteristics can be produced. Thus, for example, a pattern having anarray of adjacent wells can be created by varying thehydrophobicity/hydrophilicity, charge and other chemical characteristicsof the pattern constituents. In one embodiment, hydrophilic compoundscan be confined to individual wells by patterning walls usinghydrophobic materials. Similar substrate configurations are accessiblethrough microprinting a layer with the desired characteristics directlyonto the substrate. See, Mrkish, M.; Whitesides, G. M., Ann. Rev.Biophys. Biomol. Struct. 25:55-78 (1996).

[0129] Sequence Specificity Database

[0130] As high-resolution, high-sensitivity enzyme sequence specificityand datasets become available to the art, significant progress in theareas of diagnostics, therapeutics, drug development, biosensordevelopment, and other related areas is possible. For example, diseasemarkers can be identified and utilized for better confirmation of adisease condition or stage (see, U.S. Pat. Nos. 5,672,480; 5,599,677;5,939,533; and 5,710,007). Subcellular toxicological information can begenerated to better direct drug structure and activity correlation (see,Anderson, L., “Pharmaceutical Proteomics: Targets, Mechanism, andFunction,” paper presented at the IBC Proteomics conference, Coronado,Calif. (Jun. 11-12, 1998)). Subcellular toxicological information canalso be utilized in a biological sensor device to predict the likelytoxicological effect of chemical exposures and likely tolerable exposurethresholds (see, U.S. Pat. No. 5,811,231). Similar advantages accruefrom datasets relevant to other biomolecules and bioactive agents (e.g.,nucleic acids, saccharides, lipids, drugs, and the like).

[0131] Thus, in another preferred embodiment, the present inventionprovides a database that includes at least one set of peptide sequencespecificity data for an enzyme, preferably a protease. The datacontained in the database is acquired using a method of the inventionand/or a fluorogenic species of the invention either singly or in alibrary format. The database can be in substantially any form in whichdata can be maintained and transmitted, but is preferably an electronicdatabase. The electronic database of the invention can be maintained onany electronic device allowing for the storage of and access to thedatabase, such as a personal computer, but is preferably distributed ona wide area network, such as the World Wide Web.

[0132] The focus of the present section on databases including peptidesequence specificity data is for clarity of illustration only. It willbe apparent to those of skill in the art that similar databases can beassembled for any of the fluorogenic compounds or libraries of compoundsof the invention.

[0133] The compositions and methods described herein for identifyingand/or quantitating the relative and/or absolute abundance of a varietyof molecular and macromolecular species from a biological sample providean abundance of information, which can be correlated with pathologicalconditions, predisposition to disease, drug testing, therapeuticmonitoring, gene-disease causal linkages, identification of correlatesof immunity and physiological status, among others. As the large amountsof raw data generated by these methods are poorly suited for manualreview and analysis without prior data processing using high-speedcomputers, several methods for indexing and retrieving biomolecularinformation have been proposed. For example, U.S. Pat. Nos. 6,023,659and 5,966,712 disclose a relational database system for storingbiomolecular sequence information in a manner that allows sequences tobe catalogued and searched according to one or more protein functionhierarchies. U.S. Pat. No. 5,953,727 discloses a relational databasehaving sequence records containing information in a format that allows acollection of partial-length DNA sequences to be catalogued and searchedaccording to association with one or more sequencing projects forobtaining full-length sequences from the collection of partial lengthsequences. U.S. Pat. No. 5,706,498 discloses a gene database retrievalsystem for making a retrieval of a gene sequence similar to a sequencedata item in a gene database based on the degree of similarity between akey sequence and a target sequence. U.S. Pat. No. 5,538,897 discloses amethod using mass spectroscopy fragmentation patterns of peptides toidentify amino acid sequences in computer databases by comparison ofpredicted mass spectra with experimentally-derived mass spectra using acloseness-of-fit measure. U.S. Pat. No. 5,926,818 discloses amulti-dimensional database comprising a functionality formulti-dimensional data analysis described as on-line analyticalprocessing (OLAP), which entails the consolidation of projected andactual data according to more than one consolidation path or dimension.U.S. Pat No. 5,295,261 reports a hybrid database structure in which thefields of each database record are divided into two classes,navigational and informational data, with navigational fields stored ina hierarchical topological map which can be viewed as a tree structureor as the merger of two or more such tree structures.

[0134] The present invention provides a method for producing a computerdatabase comprising a computer and software for storing incomputer-retrievable form a collection of enzyme peptide sequencespecificity records cross-tabulated, for example, with data specifyingthe source of the protein-containing sample from which each sequencespecificity record was obtained.

[0135] In a preferred embodiment, at least one of the sources ofprotein-containing sample is from a tissue sample known to be free ofpathological disorders. In a variation, at least one of the sources is aknown pathological tissue specimen, for example, a neoplastic lesion ora tissue specimen containing an infectious agent such as a virus, or thelike. In another variation, the sequence specificity recordscross-tabulate one or more of the following parameters for each proteinspecies in a sample: (1) a unique identification code, which cancomprise a peptide sequence specificity and/or characteristic separationcoordinate (e.g., electrophoretic coordinates); (2) sample source; (3)absolute and/or relative quantity of the protein species present in thesample; (4) presence or absence of amine- or carboxy-terminalpost-translational modifications; and (5) original amino acid sequence,electrophoresis and/or mass spectral data, and the like, used toidentify the proteins.

[0136] The invention also provides for the storage and retrieval of acollection of peptide sequence specificities in a computer data storageapparatus, which can include magnetic disks, optical disks,magneto-optical disks, DRAM, SRAM, SGRAM, SDRAM, RDRAM, DDR RAM,magnetic bubble memory devices, and other data storage devices,including CPU registers and on-CPU data storage arrays. Typically, thepeptide sequence specificity records are stored as a bit pattern in anarray of magnetic domains on a magnetizable medium or as an array ofcharge states or transistor gate states, such as an array of cells in aDRAM device (e.g., each cell comprised of a transistor and a chargestorage area, which may be on the transistor). In one embodiment, theinvention provides such storage devices, and computer systems builttherewith, comprising a bit pattern encoding a protein expressionfingerprint record comprising unique identifiers for at least 10 proteinspecies cross-tabulated with sample source.

[0137] The invention preferably provides a method for identifyingrelated peptide sequences, comprising performing a computerizedcomparison between a peptide sequence specificity stored in or retrievedfrom a computer storage device or database and at least one othersequence; such comparison can comprise a sequence analysis or comparisonalgorithm or computer program embodiment thereof (e.g., FASTA, TFASTA,GAP, BESTFIT) and/or the comparison may be of the relative amount of apeptide sequence in a pool of sequences determined from a polypeptidesample of a specimen. The invention provides a computer systemcomprising a storage device having a bit pattern encoding a databasehaving at least 100 protein expression fingerprint records obtained bythe methods of the invention, and a program for sequence alignment andcomparison to predetermined genetic or protein sequences.

[0138] The invention also preferably provides a magnetic disk, such asan IBM-compatible (DOS, Windows, Windows95/98/2000, Windows NT, OS/2) orother format (e.g., Linux, SunOS, Solaris, AIX, SCO Unix, VMS, MV,Macintosh, etc.) floppy diskette or hard (fixed, Winchester) disk drive,comprising a bit pattern encoding a protein expression fingerprintrecord; often the disk will comprise at least one other bit patternencoding a polynucleotide and/or polypeptide sequence other than apeptide sequence record of the invention, typically in a file formatsuitable for retrieval and processing in a computerized sequenceanalysis, comparison, or relative quantitation method.

[0139] The invention also provides a network, comprising a plurality ofcomputing devices linked via a data link, such as an Ethernet cable(coax or 10BaseT), telephone line, ISDN line, wireless network, opticalfiber, or other suitable signal tranmission medium, whereby at least onenetwork device (e.g., computer, disk array, etc.) comprises a pattern ofmagnetic domains (e.g., magnetic disk) and/or charge domains (e.g., anarray of DRAM cells) composing a bit pattern encoding a proteinexpression fingerprint record of the invention.

[0140] The invention also provides a method for transmitting a peptidesequence specificity record of the invention that includes generating anelectronic signal on an electronic communications device, such as amodem, ISDN terminal adapter, DSL, cable modem, ATM switch, or the like,wherein the signal includes (in native or encrypted format) a bitpattern encoding a peptide sequence specificity record or a databasecomprising a plurality of peptide sequence specificity records obtainedby the method of the invention.

[0141] In a preferred embodiment, the invention provides a computersystem for comparing a query polypeptide sequence or query peptidesequence specificity to a database containing an array of datastructures, such as a peptide sequence specificity record obtained bythe method of the invention, and ranking database sequences based on thedegree of sequence identity and gap weight to the query sequence. Acentral processor is initialized to load and execute the computerprogram for alignment and/or comparison of the amino acid sequences. Aquery sequence including at least 2 amino acids or 6 nucleotidesencoding 2 amino acids is entered into the central processor via an I/Odevice. Execution of the computer program results in the centralprocessor retrieving the sequence data from the data file, whichcomprises a binary description of a peptide sequence specificity recordor portion thereof containing polypeptide sequence data for the record.

[0142] The sequence data or record and the computer program can betransferred to secondary memory, which is typically random access memory(e.g., DRAM, SRAM, SGRAM, or SDRAM). Sequences are ranked according tothe degree of sequence identity to the query sequence and results areoutput via an I/O device. For example, a central processor can be aconventional computer (e.g., Intel Pentium, PowerPC, Alpha, PA-8000,SPARC, MIPS 4400, MIPS 10000, VAX, etc.); a program can be a commercialor public domain molecular biology software package (e.g., UWGCGSequence Analysis Software, Darwin); a data file can be an optical ormagnetic disk, a data server, a memory device (e.g., DRAM, SRAM, SGRAM,SDRAM, EPROM, bubble memory, flash memory, etc.); an I/O device can be aterminal comprising a video display and a keyboard, a modem, an ISDNterminal adapter, an Ethernet port, a punched card reader, a magneticstrip reader, or other suitable I/O device.

[0143] In another preferred embodiment, the invention provides acomputer program for comparing query polypeptide sequence(s) or querypolynucleotide sequence(s) to a peptide sequence specificity databaseobtained by a method of the invention and ranking database sequencesbased on the degree of similarity of protein species expressed andrelative and/or absolute abundance in a sample. The initial step isinput of a query peptide sequence, or peptide sequence specificityrecord obtained by a method of the invention, input via an I/O device. Adata file is accessed in to retrieve a collection of peptide sequencespecificity records for comparison to the query. Individually orcollectively sequences or other cross-tabulated information of thepeptide sequence specificity collection are optimally matched to thequery sequence(s), such as by the algorithm of Needleman and Wunsch orthe algorithm of Smith and Waterman or another suitable algorithmobtainable by those skilled in the art.

[0144] Once aligned or matched, the percentage of sequence similaritycan be computed for each aligned or matched sequence to generate asimilarity value for each sequence or peptide sequence specificityrecord collection as compared to the query sequence(s). Sequences aregenerally ranked in order of greatest sequence identity or weightedmatch to the query sequence, and the relative ranking of the sequence tothe best matches in the collection of records is thus generated. Adetermination is made; if more sequences records exist in the data file,the additional sequences or a subset thereof are retrieved and theprocess is iterated. If no additional sequences exist in the data file,the rank ordered sequences are output via an I/O device, therebydisplaying the relative ranking of sequences among the sequences of thedata file optimally matched and compared to the query sequence(s).

[0145] The invention also preferably provides the use of a computersystem, such as that described above, which comprises: (1) a computer;(2) a stored bit pattern encoding a collection of peptide sequencespecificity records obtained by the methods of the invention, which maybe stored in the computer; (3) a comparison sequence, such as a querysequence; and (4) a program for alignment and comparison, typically withrank-ordering of comparison results on the basis of computed similarityvalues.

[0146] In a preferred embodiment, neural network patternmatching/recognition software is trained to identify and match peptidesequence specificity records based on backpropagation using empiricaldata input by a user. The computer system and methods described hereinpermit the identification of the relative relationship of a querypeptide sequence specificity to a collection of peptide sequencespecificities; preferably peptide sequence specificities (query anddatabase) are obtained by the methods of the invention.

[0147] The invention also provides a computer system including adatabase containing a plurality of peptide sequence specificity recordsin the form of tree-based or otherwise hierarchical navigational fieldscross-tabulated to informational data such as one or more or thefollowing: medical records, patient medical history, medical diagnostictest results of a patient, patient name, patient sex, patient age,patient genetic profile, patient diagnosis-related group code, patienttherapy, time of day, vital signs of a patient, drug assay results of apatient, medical information of patient's blood relatives, and othersimilar medical, biological, and physiological information of a patientfrom which the sample(s) used to generate the peptide sequencespecificity record was obtained.

[0148] In a preferred embodiment, a computer system comprising adatabase having a hybrid data structure with the navigational field(s)comprising a peptide sequence specificity obtained by a method of theinvention is employed to link to informational fields of the same or arelated record which comprise medical information as described herein;the data structure can conform to the general description in U.S. Pat.No. 5,295,261, which is incorporated herein by reference.

[0149] The invention also preferably provides a computer system,including a computer and a program employing a neural network trained toextract database records having a predicted or predetermined peptidesequence specificity match that is pathognomonic for a predetermineddisease or medical condition, predisposition to disease, orphysiological state. In an illustrative embodiment, a blood or cellularsample from a patient is analyzed according to a method of the inventionto provide a predetermined peptide sequence specificity that is enteredas a database query into a trained neural network that has beenpreviously trained on a plurality of predetermined database records toestablish correlative neural relationships between peptide sequencespecificity (navigation fields) and medical data (information field(s)),so that the query identifies the medical condition(s) most highlycorrelated in the trained neural network with a peptide sequencespecificity. The method can alternatively, or in addition, employ apredetermined peptide sequence specificity record obtained from serum,blood, or other cellular sample to query a database of sequencespecificity profile records using a trained neural network which linksthe query metabolite profile record to the database records linked tothe medical condition(s) most highly correlated in the trained neuralnetwork with the patient's peptide sequence specificity.

[0150] The invention also preferably provides a computer system,including a computer and a program employing a database comprisingrecords having a field or plurality of fields including, for example, apeptide sequence specificity data set obtained from a serum, blood, orother cellular sample of a patient and analyzed according to a method ofthe present invention, and further having one or a plurality of fieldscontaining data obtained from a patient relating to symptoms, medicalstatus, medical history, or other differential diagnosis information,which can be entered via a connection to the Internet or other TCP/IP orrelated networking system.

[0151] Kits

[0152] The present invention also provides for kits for the detection ofa selected species (e.g., enzyme, nucleic acid, etc.) or activity (e.g.,enzymatic, hybridization, etc.) in samples. The kits comprise one ormore containers containing the fluorogenic compounds (“indicators”) ofthe present invention. The fluorogenic compounds may be provided insolution or bound to a solid support. Thus, the kits may containindicator solutions or indicator “dipsticks”, blotters, culture media,and the like. The kits may also contain indicator cartridges (where thefluorogenic compound is bound to a solid support) for use in automatedprotease activity detectors.

[0153] The kits additionally may include an instruction manual thatteaches a method of the invention and describes the use of thecomponents of the kit. In addition, the kits may also include otherreagents, buffers, various concentrations of enzyme inhibitors, stockenzymes (for generation of standard curves, etc), culture media,disposable cuvettes and the like to aid the detection of proteaseactivity utilizing the fluorogenic protease indicators of the presentinvention.

[0154] It will be appreciated that kits may additionally, oralternatively, include any of the other indicators described herein(e.g., nucleic acid based indicators, oligosaccharide indicators, lipidindicators, etc.).

[0155] In another embodiment, the kit contains a solid support of theinvention and, optionally, directions for using the solid support forpreparing a fluorogenic compound. The kit may also contain reagents,buffers, etc. useful in preparing a fluorogenic conjugate of theinvention.

[0156] METHODS

[0157] Protease Assay

[0158] The assays of the invention are illustrated by the followingdiscussion focusing on protease assays. The focus of this discussion isfor clarity of illustration and should not be interpreted as limitingthe scope of the invention to assays of proteases. Those of skill in theart will appreciate that the broad range of compounds that can beproduced using the solid support of the present invention can be assayedusing methods known in the art or modifications on those methods thatare well within the abilities of one of skill in the art.

[0159] Proteases represent a number of families of proteolytic enzymesthat catalytically hydrolyze peptide bonds. Principal groups ofproteases include metalloproteases, serine proteases, cysteine proteasesand aspartic proteases. Proteases, in particular serine proteases, areinvolved in a number of physiological processes such as bloodcoagulation, fertilization, inflammation, hormone production, the immuneresponse and fibrinolysis.

[0160] Numerous disease states are caused by and can be characterized byalterations in the activity of specific proteases and their inhibitors.For example emphysema, arthritis, thrombosis, cancer metastasis and someforms of hemophilia result from the lack of regulation of serineprotease activities (see, for example, TEXTBOOK OF BIOCHEMISTRY WITHCLINICAL CORRELATIONS, John Wiley and Sons, Inc. N.Y. (1993)). In caseof viral infection, the presence of viral proteases have been identifiedin infected cells. Such viral proteases include, for example, HIVprotease associated with AIDS and NS3 protease associated with HepatitisC. These viral proteases play a critical role in the virus life cycle.

[0161] Proteases have also been implicated in cancer metastasis.Increased synthesis of the protease urokinase has been correlated withan increased ability to metastasize in many cancers. Urokinase activatesplasmin from plasminogen which is ubiquitously located in theextracellular space and its activation can cause the degradation of theproteins in the extracellular matrix through which the metastasizingtumor cells invade. Plasmin can also activate the collagenases thuspromoting the degradation of the collagen in the basement membranesurrounding the capillaries and lymph system thereby allowing tumorcells to invade into the target tissues (Dano, et al. Adv. Cancer. Res.,44: 139 (1985)).

[0162] Human mast cells express at least four distinct tryptases,designated α βI, βII, and βIII. These enzymes are not controlled byblood plasma proteinase inhibitors and only cleave a few physiologicalsubstrates in vitro. The tryptase family of serine proteases has beenimplicated in a variety of allergic and inflammatory diseases involvingmast cells because of elevated tryptase levels found in biologicalfluids from patients with these disorders. However, the exact role oftryptase in the pathophysiology of disease remains to be delineated. Thescope of biological functions and corresponding physiologicalconsequences of tryptase are substantially defined by their substratespecificity.

[0163] Tryptase is a potent activator of pro-urokinase plasminogenactivator (uPA), the zymogen form of a protease associated with tumormetastasis and invasion. Activation of the plasminogen cascade,resulting in the destruction of extracellular matrix for cellularextravasation and migration, may be a function of tryptase activation ofpro-urokinase plasminogen activator at the P4-P1 sequence ofPro-Arg-Phe-Lys (Stack, et al., Journal of Biological Chemistry 269(13):9416-9419 (1994)). Vasoactive intestinal peptide, a neuropeptide that isimplicated in the regulation of vascular permeability, is also cleavedby tryptase, primarily at the Thr-Arg-Leu-Arg sequence (Tam, et al., Am.J. Respir. Cell Mol Biol. 3: 27-32 (1990)). The G-protein coupledreceptor PAR-2 can be cleaved and activated by tryptase at theSer-Lys-Gly-Arg sequence to drive fibroblast proliferation, whereas thethrombin activated receptor PAR-1 is inactivated by tryptase at thePro-Asn-Asp-Lys sequence (Molino et al., Journal of Biological Chemistry272(7): 4043-4049 (1997)). Taken together, this evidence suggests acentral role for tryptase in tissue remodeling as a consequence ofdisease. This is consistent with the profound changes observed inseveral mast cell-mediated disorders. One hallmark of chronic asthma andother long-term respiratory diseases is fibrosis and thickening of theunderlying tissues that could be the result of tryptase activation ofits physiological targets. Similarly, a series of reports during thepast year have shown angiogenesis to be associated with mast celldensity, tryptase activity and poor prognosis in a variety of cancers(Coussens et al., Genes and Development 13(11): 1382-97 (1999));Takanami et al., Cancer 88(12): 2686-92 (2000); Toth-Jakatics et al.,Human Pathology 31 (8): 955-960 (2000); Ribatti et al., InternationalJournal of Cancer 85(2): 171-5 (2000)).

[0164] Tryptase has been recognized as a viable drug target, andtherapeutically useful inhibitors have been under development by severalpharmaceutical companies, some even taking advantage of the bifunctionalactive site (Burgess et al., Proceedings of the National Academy ofSciences 96(15): 8348-52 (1999); Rice et al., Curr Pharm Des 4(5):381-96 (1998)). Insights gained from the modeling of the optimalsequence into the active site will support further development of novelselective substrates of β-tryptases that will enhance our understandingof the pathophysiology of these enzymes, as well as lead to thedevelopment of new and effective inhibitors.

[0165] Clearly, measurement of changes in the activity of specificproteases is clinically significant in the treatment and management ofthe underlying disease states. Proteases, however, are not easy toassay. Typical approaches include ELISA using antibodies that bind theprotease or RIA using various labeled substrates; with their naturalsubstrates assays are difficult to perform and expensive. With currentlyavailable synthetic substrates the assays are expensive, insensitive andnonselective. In addition, many “indicator” substrates require highquantities of protease which results, in part, in the self destructionof the protease.

[0166] Thus, in a preferred embodiment, the invention provides a methodof assaying for the presence of an enzymatically active protease in asample. The method includes: (a) contacting the sample with a materialaccording to Formula II, in such a manner whereby the fluorogenic moietyis released from the peptide sequence upon action of the protease,thereby producing a fluorescent moiety; and (b) observing whether thesample undergoes a detectable change in fluorescence, the detectablechange being an indication of the presence of the enzymatically activeprotease in the sample.

[0167] The method of the invention can be used to assay forsubstantially any known or later discovered enzyme and is of particularuse in assaying for a protease. The sample containing the protease canbe derived from substantially any source, or organism. In a preferredembodiment, the sample is a clinical sample from a subject. In apresently preferred embodiment, the protease is a member selected fromthe group consisting of aspartic protease, cysteine protease,metalloprotease and serine protease. The method of the invention isparticularly preferred for the assay of proteases derived from amicroorganism, including, but not limited to, bacteria, fungi, yeast,viruses, and protozoa.

[0168] In an illustrative application, the fluorogenic molecules of thisinvention are used to assay the activity of purified protease made up asa reagent (e.g. in a buffer solution) for experimental or industrialuse. Like many other enzymes, proteases may loose activity over time,especially when they are stored as their active forms. In addition, manyproteases exist naturally in an inactive precursor form (e.g. azymogen), which itself must be activated by hydrolysis of a particularpeptide bond to produce the active form of the enzyme prior to use.Because the degree of activation is variable and because proteases mayloose activity over time, it is often desirable to verify that theprotease is active and to often quantify the activity before using aparticular protease in a particular application.

[0169] Assaying for protease activity of a stock solution simplyrequires adding a quantity of the stock solution to a fluorogenicprotease indicator of the present invention and measuring the subsequentincrease in fluorescence or decrease in excitation band in theabsorption spectrum. The stock solution and the fluorogenic indicatormay also be combined and assayed in a “digestion buffer” that optimizesactivity of the protease. Buffers suitable for assaying proteaseactivity are well known to those of skill in the art. In general, abuffer will be selected whose pH corresponds to the pH optimum of theparticular protease. For example, a buffer particularly suitable forassaying elastase activity consists of 50 mM sodium phosphate, 1 mM EDTAat pH 8.9. The measurement is most easily made in a fluorometer, andinstrument that provides an “excitation” light source for thefluorophore and then measures the light subsequently emitted at aparticular wavelength. Comparison with a control indicator solutionlacking the protease provides a measure of the protease activity. Theactivity level may be precisely quantified by generating a standardcurve for the protease/indicator combination in which the rate of changein fluorescence produced by protease solutions of known activity isdetermined.

[0170] While detection of the fluorogenic compounds is preferablyaccomplished using a fluorometer, detection may by a variety of othermethods well known to those of skill in the art. Thus, for example,since the fluorophores of the present invention emit in the visiblewavelengths, detection may be simply by visual inspection offluorescence in response to excitation by a light source. Detection mayalso be by means of an image analysis system utilizing a video camerainterfaced to a digitizer or other image acquisition system. Detectionmay also be by visualization through a filter, as under a fluorescencemicroscope. The microscope may provide a signal that is simplyvisualized by the operator. Alternatively, the signal may be recorded onphotographic film or using a video analysis system. The signal may alsosimply be quantified in realtime using either an image analysis systemor a photometer.

[0171] Thus, for example, a basic assay for protease activity of asample will involve suspending or dissolving the sample in a buffer (atthe pH optima of the particular protease being assayed), adding to thebuffer one of the fluorogenic protease indicators of the presentinvention, and monitoring the resulting change in fluorescence using aspectrofluorometer. The spectrofluorometer will be set to excite thefluorophore at the excitation wavelength of the fluorophore and todetect the resulting fluorescence at the emission wavelength of thefluorophore.

[0172] Previous approaches to verifying or quantifying protease activityinvolve combining an aliquot of the protease with its substrate,allowing a period of time for digestion to occur and then measuring theamount of digested protein, most typically by HPLC. This approach istime consuming, utilizes expensive reagents, requires a number of stepsand entails a considerable amount of labor. In contrast, the fluorogenicreagents of the present invention allow rapid determination of proteaseactivity in a matter of minutes in a single-step procedure. An aliquotof the protease to be tested is simply added to, or contacted with, thefluorogenic reagents of this invention and the subsequent change influorescence is monitored (e.g., using a fluorometer or a fluorescencemicroplate reader).

[0173] In addition to determining protease activity in “reagent”solutions, the fluorogenic compositions of the present invention may beutilized to detect protease activity in biological samples. The term“biological sample”, as used herein, refers to a sample obtained from anorganism or from components (e.g., cells) of an organism. The sample maybe of any biological tissue or fluid. Frequently the sample will be a“clinical sample” which is a sample derived from a patient. Such samplesinclude, but are not limited to, sputum, blood, blood cells (e.g., whitecells), tissue or fine needle biopsy samples, urine, peritoneal fluid,and pleural fluid, or cells therefrom. Biological samples may alsoinclude sections of tissues such as frozen sections taken forhistological purposes.

[0174] In one embodiment, the present invention provides for methods ofdetecting protease activity in an isolated biological sample. This maybe determined by simply contacting the sample with a fluorogenicprotease “indicator” of the present invention and monitoring the changein fluorescence of the “indicator” over time. The sample may besuspended in a “digestion buffer” as described above. The sample mayalso be cleared of cellular debris, e.g. by centrifugation beforeanalysis.

[0175] In another embodiment, this invention provides for a method ofdetecting in situ protease activity in histological sections. Thismethod of detecting protease activity in tissues offers significantadvantages over prior art methods (e.g. specific stains, antibodylabels, etc.) because, unlike simple labeling approaches, in situ assaysusing the protease indicators indicate actual activity rather thansimple presence or absence of the protease. Proteases are often presentin tissues in their inactive precursor (zymogen) forms which are capableof binding protease labels. Thus, traditional labeling approachesprovide no information regarding the physiological state, vis a visprotease activity, of the tissue.

[0176] The in situ assay method generally comprises providing a tissuesection (preferably a frozen section, as fixation or embedding maydestroy protease activity in the sample), contacting the section withone of the fluorogenic peptides of the present invention, andvisualizing the resulting fluorescence. Visualization is preferablyaccomplished utilizing a fluorescence microscope. The fluorescencemicroscope provides an “excitation” light source to induce fluorescenceof the fluorophore. The microscope is typically equipped with filters tooptimize detection of the resulting fluorescence. As indicated above,the microscope may be equipped with a camera, photometer, or imageacquisition system.

[0177] The fluorogenic peptide can be introduced to the sections in anumber of ways. For example, the fluorogenic peptide may be provided ina buffer solution, as described above, which is applied to the tissuesection. Alternatively, the fluorogenic peptide may be provided as asemi-solid medium such as a gel or agar which is spread over the tissuesample. The gel helps to hold moisture in the sample while providing asignal in response to protease activity. The fluorogenic peptide mayalso be provided conjugated to a polymer such as a plastic film whichmay be used in procedures similar to the development of Western Blots.The plastic film is placed over the tissue sample on the slide and thefluorescence resulting from cleaved indicator molecules is viewed in thesample tissue under a microscope.

[0178] Typically the tissue sample is incubated for a period of timesufficient to allow a protease to cleave the fluorogenic peptide.Incubation times will generally range from about 10 to 60 minutes attemperatures up to and including 37° C.

[0179] In yet another embodiment, this invention provides for a methodof detecting in situ enzymatic activity of cells in culture or cellsuspensions derived from tissues, biopsy samples, or biological fluids(e.g., saliva, blood, urine, lymph, plasma, etc.). In an illustrativeembodiment, the cultured cells are grown either on chamber slides or insuspension and then transferred to histology slides bycytocentrifugation. Similarly, the cell suspensions are preparedaccording to standard methods and transferred to histology slides. Theslide is washed with phosphate buffered saline and coated with asemi-solid polymer or a solution containing the fluorogenic proteaseindicator. The slide is incubated at 37° C. for a time sufficient for aprotease to cleave the protease “indicator”. The slide is then examinedunder a fluorescence microscope equipped with the appropriate filters,as described above.

[0180] Alternatively, the cells are incubated with the fluorogenicpeptide at 37° C., then washed with buffer and transferred to a glasscapillary tube and examined under a fluorescence microscope. When a flowcytometer is used to quantitate the intracellular enzyme activity, thecells with the fluorogenic “indicator” is simply diluted with bufferafter 37° C. incubation and analyzed.

[0181] Previously described fluorogenic protease indicators typicallyabsorb light in the ultraviolet range (e.g., Wang et al., TetrahedronLett. 31:6493 (1990)). They are thus unsuitable for sensitive detectionof protease activity in biological samples which typically containconstituents (e.g., proteins) that absorb in the ultraviolet range. Incontrast, the fluorescent indicators of the present invention bothabsorb and emit in the visible range (400 nm to about 750 nm). Thesesignals are, therefore, not readily quenched by, or otherwise interferedwith by background molecules; therefore, they are easily detected inbiological samples.

[0182] In an illustrative embodiment, the invention provides a libraryuseful for profiling of various serine and cysteine proteases. Thelibrary is able to distinguish proteases having specificity forP1-acidic amino acids (granzyme B), P1-large hydrophobic (chymotrypsin),P1-small hydrophobic (human neutrophil elastase), P1-basic amino acids(trypsin, thrombin, plasmin) and P1-multiple amino acids (papain andcruzain) (FIG. 2).

[0183] In another illustrative embodiment, the invention provides alibrary for probing the extended substrate specificity of several serineproteases involved in blood coagulation, in which the P1 position isheld constant as either Lys or Arg, depending on the preferredP1-specificity of the protease. Thrombin, plasmin, uPA, tPA and factorXa (FIG. 3A-E) display profiles consistent with knowledge about theirspecificity.

[0184] The invention also provides a library for probing the extendedsubstrate specificity of the cysteine proteases, papain and cruzain,having P1-positioned libraries including peptides having hydrophobicamino acids in the P2 position.

[0185] The PS-SCL strategy provided by the present invention allows forthe rapid and facile determination of proteolytic substrate specificity.Those of skill in the art will appreciate that the present inventionprovides a wide variety of alternative library formats. For example,fixing the P2-position as a large hydrophobic amino acid may circumventpreferential internal cleavage by papain-fold proteases and lead toproper register of the substrate. Determination and consideration ofparticular limitations relevant to any particular enzyme or method ofsubstrate specificity determination are within the ability of those ofskill in the art.

[0186] In addition to its use in assaying for the presence of a selectedenzyme, the method of the invention is also useful for detecting,identifying and quantifying an enzyme (e.g., protease). Thus, in anotherpreferred embodiment, the method further includes, (c) quantifying thefluorescent moiety, thereby quantifying the protease.

[0187] In yet another preferred embodiment, the invention provides amethod of assaying for the presence of an enzyme, for example, anenzymatically active protease in a sample using a peptide of theinvention having a structure according to Formula VI. The methodincludes: (a) contacting the sample with a material according to FormulaVI, in such a manner whereby the fluorogenic moiety is released from thepeptide sequence upon action of the protease, thereby producing afluorescent moiety; and (b) observing whether the sample undergoes adetectable change in fluorescence, the detectable change being anindication of the presence of the enzymatically active protease in thesample. Preferred embodiments of this method are substantially similarto those set forth for the method using the material according toFormula II.

[0188] In a preferred embodiment of the above-described method, themethod further includes, (d) quantifying the fluorescent moiety, therebyquantifying the protease.

[0189] Protease Sequence Specificity Assay

[0190] In another preferred embodiment, the present invention provides amethod of determining the sequence specificity of an enzyme, andpreferably of an enzymatically active protease. The method includes: (a)contacting the protease with a library of peptides of the invention insuch a manner whereby the fluorogenic moiety is released from thepeptide sequence, thereby forming a fluorescent moiety; (b) detectingthe fluorescent moiety; and (c) determining the sequence of the peptidesequence, thereby determining the peptide sequence specificity profileof the protease.

[0191] In a preferred embodiment of the above-described method, themethod further includes, (d) quantifying the fluorescent moiety, therebyquantifying the protease.

[0192] Microorganism Assay

[0193] In a further preferred embodiment, the invention provides amethod of assaying for the presence of a selected microorganism in asample by probing the sequence specificity of an enzyme or othermolecule produced or utilized by the microorganism. In an illustrativeembodiment, the enzyme is a protease, which mediates peptide cleavage bythe microorganism of one or more peptides of the invention. The methodincludes: (a) contacting a sample suspected of containing the selectedmicroorganism with a material according to Formula VII, wherein thepeptide comprises a sequence that is selectively cleaved by a proteaseof the selected microorganism, thereby releasing the fluorogenic moietyfrom the peptide sequence; and (b) detecting the cleavage by detectingfluorescence arising from a fluorescent moiety produced by cleavage ofthe fluorogenic moiety from the peptide sequence, thereby confirming thepresence of the selected microorganism in the sample. The preferredembodiments of the present method are substantially similar to thosedescribed in conjunction with the protease assay, supra.

[0194] In yet another preferred embodiment, the invention provides amethod of assaying for the presence of a selected microorganism in asample by probing the sequence specificity of peptide cleavage by aprotease of the microorganism using a peptide of the invention having astructure according to Formula VII. The method includes: (a) contactinga sample suspected of containing the selected microorganism with apeptide according to Formula VII. The peptide comprises a sequence thatis selectively cleaved by a protease of a selected microorganism,thereby releasing the fluorogenic moiety from the peptide sequence; and(b) detecting the cleavage by detecting fluorescence arising from afluorescent moiety produced by cleavage of the fluorogenic moiety fromthe peptide sequence, thereby confirming the presence of the selectedmicroorganism in the sample.

[0195] In a preferred embodiment of the above-described method, themethod further includes, (d) quantifying the fluorescent moiety, therebyquantifying the protease, the microorganism or both.

[0196] The above-described method is useful to determine whether anunknown microorganism contains an enzyme that acts on a peptide of theinvention to liberate a fluorescent moiety, and it may be include withinor utilized in conjunction with a device in which identification of anunknown microorganism is made on the basis of its enzyme content (see,for example, Mize, U.S. Pat No. 5,055,594).

[0197] The methods of the invention are also useful for determining theeffect of an agent, such as an antimicrobial agent on a microorganism.Thus, the invention can, for example, take the form of a process fordetermining the minimum inhibitory concentration (MIC) of anantimicrobial substance with respect to a microorganism under study(e.g., a clinical septic isolate). In an illustrative embodiment, amicroorganism is treated with an antimicrobial agent that inhibits ordestroys an enzyme or other molecule necessary for the growth and/orreproduction of the organism. The effect of the antimicrobial agent onthe microorganism is probed by contacting the microorganism with one ormore of the fluorogenicpeptides of the invention. A change in theability of the enzyme of the microorganism to produce a fluorescentmaterial from the fluorogenic peptide is indicative of the activity ofthe antimicrobial agent. The magnitude of the effect, can be ascertainedby quantitating the fluorescence and comparing it to a selectedbenchmark, such as the magnitude of fluorescence arising from contactingthe microorganism with a peptide of the invention in the absence of anantimicrobial agent (see, for example, Carr et al, U.S. Pat. No.5,064,756, and U.S. Pat No. 5,079,144).

[0198] In the above-recited methods, the exposure to the fluorogenicpeptide to the microorganisms lasts for a sufficient time to let theenzymatic reaction take place. The fluorescence of each sample isassessed (e.g., by a non-destructive instrumental fluorometric orfluoroscopic method).

[0199] Moreover, in each of the aspects and embodiments set forthhereinabove, the protease can be substantially any protease of interest,but is preferably a member selected from the group consisting ofaspartic protease, cysteine protease, metalloprotease and serineprotease. The protease assayed using a method of the invention can bederived from substantially any organism, including, but not limited tomammals, birds, reptiles, insects, plants, fungi and the like. In apreferred embodiment, the protease is derived from a microorganism,including, but not limited to, bacteria, fungi, yeast, viruses, andprotozoa.

[0200] Fluorogenic Peptide Synthesis

[0201] Those of skill in the art will recognize that many methods can beused to prepare the peptides and the libraries of the invention. In anexemplary embodiment (see, FIG. 1), the fluorogenic leaving group of theinvention is synthesized by condensing an N-Fmoc coumarin derivative 2,to acid-labile Rink linker to provide ACC resin 3. After Fmoc-removal toproduce free amine 4, natural, unnatural and modified amino acids can becoupled to the aniline efficiently to produce 5, which can be elaboratedby the coupling of additional amino acids to form 6, for example. Afterthe synthesis of the peptide is complete, the peptide-fluorogenic moietyconjugate can be cleaved from the solid support to form 7 or,alternatively, the conjugate can remain tethered to the solid support.

[0202] Thus, in a further preferred embodiment, the present inventionprovides a method of preparing a fluorogenic peptide or a materialincluding a fluorogenic peptide. The method includes: (a) providing afirst conjugate comprising a fluorogenic moiety covalently bonded to asolid support, the conjugate having a structure according to Formula Iwherein, at least one of R¹, R², R³, R⁴, R⁵ and R⁶ is —NH₂; (b)contacting the first conjugate with a first protected amino acid moiety(pAA¹) and an activating agent, thereby forming a peptide bond between acarboxyl group of pAA¹ and the aniline nitrogen of the first conjugate;(c) deprotecting the pAA¹, thereby forming a second conjugate having areactive AA¹ amine moiety; (d) contacting the second conjugate with asecond protected amino acid (pAA²) and an activating agent, therebyforming a peptide bond between a carboxyl group of pAA2 and the reactiveAA¹ amine moiety; and (e) deprotecting the pAA², thereby forming a thirdconjugate having a reactive AA² amine moiety.

[0203] In a preferred embodiment, the method further includes: (f)contacting the third conjugate with a third protected amino acid (pAA³)and an activating agent, thereby forming a peptide bond between acarboxyl group of pAA³ and the reactive AA² amine moiety; and (e)deprotecting the pAA³, thereby forming a fourth conjugate having areactive AA³ amine moiety.

[0204] For amino acids that are difficult to couple (Ile, Val, etc),free, unreacted aniline may remain on the support and complicatesubsequent synthesis and assay operations. A specialized capping stepemploying the 3-nitrotriazole active ester of acetic acid in DMFefficiently acylates the remaining aniline. The resulting aceticacid-capped coumarin that may be present in unpurified substratesolutions is generally not a protease substrate. P1-substituted resinsthat are provided by these methods can be used to prepare anyACC-fluorogenic substrate.

[0205] Thus, in yet another preferred embodiment, the method furtherincludes, between steps (b) and (c), capping substantially all of theaniline amine groups that have not reacted with pAA¹. The capping stepcan use any reagent system that includes an amine-reactive component. Ina preferred embodiment, the capping step utilizes a mixture comprisingan active ester of a carboxylic acid, such as, for example, thenitrotriazole ester of acetic acid.

[0206] In a further preferred embodiment, diversity at any particularposition or combination of positions is introduced by utilizing amixture of at least two, preferably at least 6, more preferably at least12 and more preferably still, at least 20, amino acids to grow thepeptide chain. Thus, a member selected from the group consisting ofpAA¹, pAA², pAA³ and combinations thereof includes a mixture ofprotected amino acids differing in the identity of the amino acidportion of the protected amino acids. The mixtures of amino acids caninclude of any useful amount of a particular amino acid in combinationwith any useful amount of one or more different amino acids. In apresently preferred embodiment, the mixture is an isokinetic mixture ofamino acids.

[0207] Solid phase peptide synthesis in which the C-terminal amino acidof the sequence is attached to an insoluble support followed bysequential addition of the remaining amino acids in the sequence is thepreferred method for preparing the peptide backbone of the compounds ofthe present invention. Techniques for solid phase synthesis aredescribed by Barany and Merrifield, Solid-Phase Peptide Synthesis; pp.3-284 in The Peptides: Analysis, Synthesis, Biology. Vol. 2; SPECIALMETHODS IN PEPTIDE SYNTHESIS, Part A., Gross and Meienhofer, eds.Academic press, N.Y., 1980; and Stewart et al., SOLID PHASE PEPTIDESYNTHESIS, 2nd ed. Pierce Chem. Co., Rockford, Ill. (1984) which areincorporated herein by reference. Solid phase synthesis is most easilyaccomplished with commercially available peptide synthesizers utilizingFmoc or t-BOC chemistry. The chemical synthesis of the peptide componentof a fluorogenic protease indicator is described in detail in Examples3, 4 and 5.

[0208] In a particularly preferred embodiment, peptide synthesis isperformed using Fmoc synthesis chemistry. The side chains of Asp, Ser,Thr and Tyr are preferably protected using t-butyl and the side chain ofCys residue using S-trityl and S-t-butylthio, and Lys residues arepreferably protected using t-Boc, Fmoc and 4-methyltrityl for lysineresidues. Appropriately protected amino acid reagents are commerciallyavailable or can be prepared using art-recognized methods. The use ofmultiple protecting groups allows selective deblocking and coupling of afluorophore to any particular desired side chain. Thus, for example,t-Boc deprotection is accomplished using TFA in dichloromethane. Fmocdeprotection is accomplished using, for example, 20% (v/v) piperidine inDMF or N-methylpyrolidone, and 4-methyltrityl deprotection isaccomplished using, for example, 1 to 5% (v/v) TFA in water or 1% TFAand 5% triisopropylsilane in DCM. S-t-butylthio deprotection isaccomplished using, for example, aqueous mercaptoethanol (10%). Removalof t-butyl, t-boc and S-trityl groups is accomplished using, forexample, TFA:phenol:water:thioanisol:ethanedithiol (85:5:5:2.5:2.5), orTFA:phenol:water (95:5:5). Detailed synthesis, deprotection andfluorophore coupling protocols are provided in the Examples herein.

[0209] The materials and methods of the present invention are furtherillustrated by the examples which follow. These examples are offered toillustrate, but not to limit the claimed invention.

EXAMPLES

[0210] Materials and Methods

[0211] Reagents and General Methods

[0212] Rink Amide AM resin and Fmoc-amino acids were purchased fromNovabiochem (San Diego, Calif.). The amine substitution level of theRink resin (0.80 meq/gram) determined by a spectrophotometricFmoc-quantitation assay (Bunin, B. A., (1998) The Combinatorial Index(Academic Press, San Diego). Anhydrous DMF, EM Science (Hawthorne,N.Y.). HATU was purchased from Perseptive Biosystems (Foster City,Calif.). DICI, HOBt, AcOH, Fmoc-Cl, TFA, collidine, and TIS werepurchased fromAldrich (Milwaukee, Wis.). Argonaut Quest 210 OrganicSynthesizer was used to prepare Fmoc-P1-substituted ACC resins. Librarysynthesis was performed in 96-well plates using the Multi-Chem synthesisapparatus of Robbins Scientific (Sunnyvale, Calif.). Human thrombin,plasmin, and factor Xa were used as received, and were purchased fromHaematologic Technologies Inc. (Essex Jct., Vt.). Human light chain uPA,and neutrophil elastase were used as received, and were purchased fromCalbiochem (San Diego, Calif.). Rat granzyme B was expressed andpurified as described (Harris, J. L., et al., (1998) Journal ofBiological Chemistry 273:27364-73). Cruzain was expressed and purifiedas described (Eakin, A. E., et al., (1992) Journal of BiologicalChemistry 267:7411-20). Rat trypsin was expressed and purified asdescribed (Halfon, S., et al., (1996) Journal of the American ChemicalSociety 118:1227-1228). DNA-modifying enzymes were obtained from Promega(Madison, Wis.). The Pichia pastoris expression system was purchasedfrom Invitrogen (San Diego, Calif.). Native human lung tryptase waspurchased from ICN (Aurora, Ohio). Factor Xa was purchased from NewEngland Biolabs (Beverly, Mass.). tPA and uPA were purchased fromAmerican Diagnostica (Greenwich, Conn.). Heparin and other biochemicalswere purchased from Sigma. Substrates in the positional scanningsynthetic combinatorial libraries as well as the single substratesAc-PRNK-ACC, Ac-PANK-ACC, PRTK-ACC, Ac-PRNR-ACC, Ac-GTAR-ACC,Ac-QFAR-ACC, Ac-KQWK-ACC, and Ac-nTPR-ACC were prepared as previouslydescribed (9). Ac-PRNK-cmk was synthesized by Enzyme Systems Products(Livermore, Calif.).

Example 1

[0213] This Example sets forth the synthesis of7-Fmoc-aminocoumarin-4-acetic acid, a precursor to the solid support ofthe invention.

[0214] 1.1 ACC-Resin Synthesis

[0215] 1.1a Synthesis of 7-Fmoc-aminocoumarin-4-acetic acid

[0216] 7-Fmoc-aminocoumarin-4-acetic acid was prepared by treating7-aminocoumarin-4-acetic acid (14, 15) with Fmoc-Cl.7-aminocoumarin-4-acetic acid (10.0 g, 45.6 mmol) and H₂O (228 mL) weremixed. NaHCO₃ (3.92 g, 45.6 mmol) was added in small portions followedby the addition of acetone (228 mL). The solution was cooled with an icebath, and Fmoc-Cl (10.7 g, 41.5 mmol) was added with vigorous stirringover the course of an hour. The ice bath was removed and the solutionstirred overnight. The acetone was removed with rotary evaporation andthe resulting gummy solid was collected via filtration and washed withseveral portions of hexane. The material was dried over P₂O₅ to give14.6 g (80%) of cream-colored solid: ¹H NMR (400 MHz) δ 3.86 (s, 2),4.33 (t, 1, J=6.2), 4.55 (d, 2, J=6.2), 6.34 (s, 1), 7.33-7.44 (m, 5),7.56 (s, 1), 7.61 (d, 1, J=8.6), 7.76 (d, 2, J=7.3), 7.91 (d, 2, J=7.4),10.23 (s, 1), 12.84 (s, 1); ¹³C (101 MHz) δ 37.9, 47.4, 66.8, 67.2,105.5, 114.6, 115.3, 121.1, 125.9, 126.9, 128.0, 128.6, 141.6, 143.6,144.5, 150.7, 154.1, 154.8, 160.8, 171.4.

Example 2

[0217] Example 2 sets forth an illustrative synthesis of a solid supportof the invention and the functionalization of the solid support with asingle amino acid residue.

[0218] 2.1 Synthesis of ACC Resin

[0219] ACC-resin was prepared by condensation of Rink Amide AM resinwith 7-Fmoc-aminocoumarin-4-acetic acid. Rink Amide AM resin (21 g, 17mmol) was solvated with DMF (200 mL). The mixture was agitated for 30min and filtered with a filter cannula (Pharmacia, Uppsala, Sweden)whereupon 20% piperidine in DMF (200 mL) was added. After agitating 25min, the resin was filtered and washed with DMF (3×200 mL).7-Fmoc-aminocoumarin-4-acetic acid (15 g, 34 mmol), HOBt (4.6 g, 34mmol), and DMF (150 mL) were added, followed by the addition of DICI(5.3 mL, 34 mmol). The mixture was agitated overnight, filtered, washed(DMF: 3×200 mL, THF: 3×200 mL, MeOH: 3×200 mL), and dried over P₂O₅. Thesubstitution level of the resin was 0.58 mmol/g (>95%) as determined byFmoc-analysis (Bunin, B. A., (1998) The Combinatorial Index (AcademicPress, San Diego).

[0220] 2.2 Synthesis of P1-Substituted ACC-Resin Synthesis

[0221] Fmoc-ACC-Resin (100 mg, 0.058 mmol) was added to 20 reactionvessels of an Argonaut Quest 210 Organic Synthesizer and solvated withDMF (2 mL). The resin was filtered and 20% piperidine in DMF (2 mL) wasadded to each vessel. After agitating for 25 min, the resin was filteredand washed with DMF (3×2 mL). An Fmoc-amino acid (0.29 mmol), DMF (0.7mmol), collidine (76 μL, 0.58 mmol) and HATU (110 mg, 0.29 mmol) wereadded to the designated reaction vessel followed by agitation for 20 h.The resins were then filtered, washed with DMF (3×2 mL), and subjected asecond time to the coupling conditions. A solution of AcOH (40 μL, 0.70mmol), DICI (110 μL, 0.70 mmol), nitrotriazole (80 mg, 0.70 mmol) in DMF(0.7 mL) was added to each of the reaction vessels followed by agitationover a 24 h period. The resins were filtered, washed (DMF: 3×2 mL; THF:3×2 mL; MeOH: 3×2 mL), and dried over P₂O₅. The substitution level ofeach resin^(‡) was determined by Fmoc-analysis (Bunin, B. A., (1998) TheCombinatorial Index (Academic Press, San Diego).

Example 3

[0222] Example 3 sets forth the synthesis and screening of libraries ofthe invention.

[0223] 3.1 P1-Diverse Library

[0224] 3.1a Synthesis

[0225] Individual P1-substituted Fmoc-amino acid ACC-resin (ca. 25 mg,0.013 mmol) was added to wells of a Multi-Chem 96-well reactionapparatus. The resin-containing wells were solvated with DMF (0.5 mL). A20% piperidine in-DMF solution (0.5 mL) was added followed by agitationfor 30 min. The wells of the reaction block were filtered and washedwith DMF (3×0.5 mL). In order to introduce the randomized P2 position,an isokinetic mixture (Ostresh, J. M., et al., (1994) Biopolymers34:1681-9) of Fmoc-amino acids (4.8 mmol, 10 equiv/well; Fmoc-aminoacid, mol%: Fmoc-Ala-OH, 3.4; Fmoc-Arg(Pbf)-OH, 6.5; Fmoc-Asn(Trt)-OH,5.3; Fmoc-Asp(O-t-Bu)-OH, 3.5; Fmoc-Glu(O-t-Bu)-OH, 3.6;Fmoc-Gln(Trt)-OH, 5.3; Fmoc-Gly-OH, 2.9; Fmoc-His(Trt)-OH, 3.5;Fmoc-Ile-OH, 17.4; Fmoc-Leu-OH, 4.9; Fmoc-Lys(Boc)-OH, 6.2; Fmoc-Nle-OH,3.8; Fmoc-Phe-OH, 2.5; Fmoc-Pro-OH, 4.3; Fmoc-Ser(O-t-Bu)-OH, 2.8;Fmoc-Thr(O-t-Bu)-OH, 4.8; Fmoc-Trp(Boc)-OH, 3.8; Fmoc-Tyr(O-t-Bu)-OH,4.1; Fmoc-Val-OH, 11.3) was pre-activated with DICI (390 μL, 2.5 mmol),and HOBt (340 mg, 2.5 mmol) in DMF (10 mL). The solution (0.5 mL) wasadded to each of the wells. The reaction block was agitated for 3 h,filtered, and washed with DMF (3×0.5 mL). The randomized P3 and P4positions were incorporated in the same manner. The Fmoc of the P4 aminoacid was removed and the resin was washed with DMF (3×0.5 mL), andtreated with 0.5 mL of a capping solution of AcOH (150 μL, 2.5 mmol),HOBt (340 mg, 2.5 mmol) and DICI (390 μL, 2.5 mmol) in DMF (10 mL).After 4 h of agitation, the resin was washed with DMF (3×0.5 mL), CH₂Cl₂(3×0.5 mL), and treated with a solution of 95:2.5:2.5 TFA/TIS/H₂O. Afterincubating for 1 h the reaction block was opened and placed on a 96deep-well titer plate and the wells were washed with additional cleavagesolution (2×0.5 mL). The collection plate was concentrated, and thesubstrate-containing wells were diluted with EtOH (0.5 mL) andconcentrated twice. The contents of the individual wells werelyophilized from CH₃CN:H₂O mixtures. The total amount of substrate ineach well was conservatively estimated to be 0.0063 mmol (50%) basedupon yields of single substrates.

[0226] 3.1b Enzymatic Assay of Library

[0227] The concentration of proteolytic enzymes was determined byabsorbance measured at 280 nm (Gill, S. C., et al., (1989) Anal Biochem182:319-26). The proportion of catalytically active thrombin, plasmin,trypsin, uPA, tPA, and chymotrypsin was quantitated by active-sitetitration with MUGB or MUTMAC (Jameson, G. W., et al., (1973)Biochemical Journal 131:107-117).

[0228] Substrates from the PS-SCLs were dissolved in DMSO. Approximately1.0×10⁻⁹ mol of each P1-Lys, P1-Arg, or P1-Leu sub-library (361compounds) was added to 57 wells of a 96-well microfluor plate (DynexTechnologies, Chantilly, Va.) for a final concentration of 0.1 μM.Approximately 1.0×10⁻¹⁰ mol of each P1-diverse sub-library (6859compounds) was added to 20 wells of a 96-well plate for a finalconcentration of 0.01 μM in each compound. Hydrolysis reactions wereinitiated by the addition of enzyme (0.02 nM-100 nM) and monitoredfluorometrically with a Perkin Elmer LS50B Luminescence Spectrometer,with excitation at 380 nm and emission at 450 nm or 460 nm. Assays ofthe serine proteases were performed at 25° C. in a buffer containing 50mM Tris, pH 8.0, 100 mM NaCl, 0-5 mM CaCl₂, 0.01% Tween-20, and 1% DMSO(from substrates). Assay of the cysteine proteases, papain and cruzain,was performed at 25° C. in a buffer containing 100 mM sodium acetate, pH5.5, 100 mM NaCl, 5 mM DTT, 1 mM EDTA, 0.01% Brij-35, and 1% DMSO (fromsubstrates).

[0229] 3.2 Results

[0230] 3.2a Profiling Proteases with a P1-Diverse Library of 137,180Substrates

[0231] To test the possibility of attaching all amino acids to theP1-site in the substrate a P1-diverse tetrapeptide library was created.The P1-diverse library consists of 20 wells in which only theP1-position is systematically held constant as all amino acids,excluding cysteine and including norleucine. The P2, P3, and P4positions consist of an equimolar mixture of all amino acids for a totalof 6,859 substrate sequences per well. Several serine and cysteineproteases were profiled to test the applicability of this library forthe identification of the optimal P1 amino acid. Chymotrypsin showed theexpected specificity for large hydrophobic amino acids (FIG. 2A).Trypsin and thrombin showed preference for P1-basic amino acids(Arg>Lys) (FIGS. 2B and 2C). Plasmin also showed a preference for basicamino acids (Lys>Arg) (FIG. 2D). Granzyme B, the only known mammalianserine protease to have P1-Asp specificity, showed a distinct preferencefor aspartic acid over all other amino acids, including the other acidicamino acid, Glu (FIG. 2E). The P1-profile for human neutrophil elastasehas the canonical preference for alanine and valine (FIG. 2F). Thecysteine proteases, papain (FIG. 2G) and cruzain (FIG. 2H) showed thebroad P1-substrate specificity that is known for these enzymes, althoughthere is a modest preference for arginine.

Example 4

[0232]4.1 P1-Fixed Library

[0233] 4.1a Synthesis

[0234] Multi-gram quantities of P1-substituted ACC-resin weresynthesized using the methods described herein. Three libraries with theP1-position fixed as Lys, Arg, or Leu were prepared. Fmoc-aminoacid-substituted ACC resin (ca. 25 mg, 0.013 mmol, of Lys, Arg, or Leu)was placed in 57 wells of a 96-well reaction block: 3 sublibrariesdenoted by the second fixed position (P4, P3, P2) of 19 amino acids(cysteine was omitted and norleucine was substituted for methionine).Synthesis, capping and cleavage of the substrates were identical to thatdescribed in the previous section, with the exception that for P2, P3and P4 sublibraries, individual amino acids, rather than isokineticmixtures, were added to the spatially-addressed P2, P3 or P4 positions.

[0235] 4.2 Results

[0236] 4.2a Profiling of Serine Proteases with P1-Fixed PositionalLibraries

[0237] The extended P4-P2 substrate specificity of several serineproteases was profiled with tetrapeptide libraries in which theP1-position was held constant. Three sub-libraries denoting the secondfixed position (P4, P3, P2) and consisting of 19 wells addressing afixed amino acid (Cys was omitted and Nle was substituted for Met) werescreened (361 compounds/well and 6,859 compounds/library). Because ofthe enhanced fluorescence properties of the ACC fluorophore, theconcentration of each substrate could be reduced to 0.1 μM, versus 0.25μM for the AMC substrates (Backes, B. J., et al., (2000) NatureBiotechnology 18:187-193).

[0238] Plasmin, a protease involved in fibrinolysis, has a P1-preferencefor lysine. Recently, we have shown plasmin to have a distinctpreference for aromatic amino acids at the P2 position and lysine at P4(Backes, B. J., et al., (2000) Nature Biotechnology 18:187-193). As isconsistent with that data, the substrate specificity profile of plasminin the ACC P1-fixed lysine library is for P4-lysine, broadP3-specificity, and P2-aromatic amino acids (FIG. 3A).

[0239] Thrombin prefers cleavage after P1-arginine to cleavage afterP1-lysine. However, the specificity preference of thrombin, whenprofiled with both the P1-Arg and P1-Lys libraries, shows littledifference in the extended subsites (FIG. 3B and Backes, B. J., et al.,(2000) Nature Biotechnology 18:187-193). Thrombin has a preference foraliphatic amino acids at the P4 position, little preference at P3, andstrict preference for proline at the P2-position. Correlation ofthrombin's optimal substrate sequence with that found in itsphysiological substrates has been noted in previous work from this lab(Backes, B. J., et al., (2000) Nature Biotechnology 18:187-193).

[0240] Two enzymes that have been extensively characterized for theirextended specificity are tissue-type plasminogen activator (t-PA) (Ding,L., et al., (1995) Proceedings of the National Academy of Sciences ofthe United States of America 92:7627-31; Coombs, G. S., et al., (1996)Journal of Biological Chemistry 271:4461-7) and urokinase plasminogenactivator (u-PA) (Ke, S. H., et al., (1997) Journal of BiologicalChemistry 272:16603-9; Ke, S. H., et al., (1997) Journal of BiologicalChemistry 272:20456-62). Both t-PA and u-PA are responsible forconverting plasminogen into active plasmin, and both show highspecificity for cleavage after P1-Arg. We observe that both enzymes alsoshow similar preference for small amino acids at P2 (Gly/Ala/Ser) and nosignificant preference at P4, except for the low activity of acidicamino acids (FIGS. 3C and 3D). In contrast, their P3 preferences arequite disparate with t-PA showing preference for aromatic amino acids(Phe and Tyr) and u-PA for small polar amino acids (Thr and Ser). Thisdifference in P3-specificity was also noted by Ke et al. to be a majordistinction between the two-plasminogen activators (Ke, S. H., et al.,(1997) Journal of Biological Chemistry 272:16603-9).

[0241] Factor Xa is an enzyme that plays the critical physiologicalfunctions of activating prothrombin and factor VII in the bloodcoagulation cascade (Davie, E. W., et al., (1991) Biochemistry30:10363-70). Through profiling with the P1-Arg library, we find factorXa to show a minor preference for P4-aliphatic amino acids, broadsubstrate specificity in P3, with the absence of P3-proline activity,and a P2-preference for glycine (FIG. 3E). This quantitative informationagrees with the qualitative sequences that are efficiently hydrolyzed byfactor Xa in a substrate-phage system (2) as well as kinetic studies ontripeptide para-nitroanilide (Cho, K., et al., (1984) Biochemistry23:644-50) and AMC substrates (Cho, K., et al., (1984) Biochemistry23:644-50; Lottenberg, R., et al., (1981) Methods in Enzymology 80 PtC:341-61). Furthermore, the factor Xa P4-P1 cleavage sequence determinedhere is found in physiologically relevant substrates: the cleavagesequences in prothrombin are Ile-Glu-Gly-Arg and Ile-Asp-Gly-Arg;cleavage sequence in factor VII is Pro-Gln-Gly-Arg; and the cleavagesequence in the autolysis loop of factor Xa is Glu-Lys-Gly-Arg(Brandstetter, H., et al., (1996) Journal of Biological Chemistry271:29988-92).

[0242] 4.2b Profiling of Cysteine Proteases with P1-Fixed PositionalLibraries

[0243] The positional substrate libraries with the ACCfluorogenic-leaving group are also conducive for defining cysteineprotease specificity. The P4-P2 extended substrate specificity forpapain and cruzain were defined using the ACC P1-fixed arginine orleucine library. Cysteine proteases of the papain-like class have beenshown to have primary substrate specificity at the P2-position(Rawlings, N. D., et al., (1994) Methods in Enzymology 244:461-86)rather than the P1-position as is seen in the chymotrypsin-like class ofserine proteases. The P2-position usually shows a preference forhydrophobic amino acids. Indeed, we observe papain to have a preferencefor P2-Val>Phe>Tyr>Nle (FIG. 3F) and cruzain to have a P2-preference forLeu>Tyr>Phe>Val (FIG. 3G). While the P3 specificity is rather broad,papain does show a preference for Pro, whereas cruzain has a preferencefor the basic amino acids, arginine and lysine. The P4 position is verybroad for both enzymes, but interesting observations arise from testingall possible substrates. There is a lack of activity for large aliphaticand aromatic amino acids, the exact amino acids that are preferred inthe P2 library. This absence is also seen in a P4 library in which theP1-position is held constant as leucine (FIG. 3H). One possible reasonfor the observations in the P4 library is that the tetrapeptidesubstrates are out of register. Cleavage is not occurring at theP1-amido-carbamoylmethyl-coumarin bond, but rather, at the P3-P2 amidebond because the large hydrophobic P4-amino acid binds to the S2-pocketof the enzyme. Incubation of the single substrate Ac-Leu-Thr-Phe-Lys-ACCwith cruzain and analysis of the cleavage products confirmed thisobservation. Product fragments corresponding to cleavage between Thr-Phewere observed (data not shown).

Example 5

[0244] Example 5 sets forth the synthesis of single peptide substratesand the kinetic assay of these substrates.

[0245] 5.1 Single Substrates.

[0246] 5.1a. Synthesis

[0247] Single substrates for kinetic analysis were prepared employingthe methods described in the Examples above. The unpurified productswere subjected to reversed-phase HPLC preparatory chromatographyfollowed by lyophilization.

[0248] 5.1b Single Substrate Kinetic Assays

[0249] Thrombin concentration ranged from 5-20 nM. The finalconcentration of substrate ranged from 0.005-2 mM, the concentration ofDMSO in the assay was less than 5%. Hydrolysis of AMC and ACC substrateswas monitored fluorometrically with an excitation wavelength of 380 nmand emission wavelength of 460 nm on a Fluoromax-2 spectrofluorometer.Cruzain (10 nM) was incubated with 600 μM of the Ac-Leu-Thr-Phe-Lys-ACCsubstrate. Aliquots were removed at various time points and applied to aC-18 reverse-phase HPLC column with a 10-40% gradient of 95:4.9:0.1Acetonitrile:H₂O:TFA. MALDI (PE Biosystems Voyager) mass spectrometrydata was collected on the HPLC fractions.

Example 6

[0250] Example 6 sets forth an experiment designed to investigate theproperties of ACC and the overlap of these properties with those of AMC.

[0251] 6.1 Fluorescence Properties of 7-Amino-4-Carbamoylmethyl-coumarin

[0252] The fluorescence of free ACC and peptidyl-derivatized ACC wasdetected on a Spex fluorometer thermostated to 25° C. Excitationwavelengths of 300-410 nm, 5 nm intervals, were used with emissionwavelengths of 410-500 nm, 5 nm intervals, to determine optimalexcitation and emission parameters.

[0253] 6.2 Results

[0254] 6.2a Fluorescence Properties of7-amino-4-carbamoylmethyl-coumarin

[0255] The excitation and emission maxima of the amino-conjugated7-amino-4-carbamoylmethyl-coumarin (ACC) substrates are 325 nm and 390nm, respectively (Table I). Cleavage of the substrate by a protease torelease the free 7-amino-4-carbamoylmethyl-coumarin results in a shiftof the excitation and emission maxima to 350 nm and 450 nm, respectively(Table I). The ACC fluorophore has an approximately 2.8-fold higherfluorescence yield than the AMC coumarin at the excitation and emissionwavelengths of 380 nm and 460 nm (Table I). The enhanced fluorescence ofthe ACC group allows for the more sensitive detection of proteolyticactivity. TABLE I λ_(max,ex) λ_(max,em) Compound (nm) (nm) RFU/nM₁RFU/nM₂ 7-Amino-4-Carbamoylmethyl- 350 450 5750 4390 coumarin (ACC)7-Nle-Thr-Pro-Lys-ACC 325 400 6.4 4.6 7-Amino-4-Methylcoumarin 340 4402600 1550 (AMC) 7-Nle-Thr-Pro-Lys-AMC 330 390 3.3 2.2

[0256] 6.2b Proteolytic Comparison of ACC and AMC

[0257] To evaluate ACC as a proteolytic leaving group, matchedtetrapeptide substrates were made that differed only in the leavinggroup, ACC or the traditionally used AMC. The two thrombin-susceptiblesequences with ACC or AMC, P4-Nle-P3-Thr-P2-Pro-P1-Lys andP4-Leu-P3-Gly-P2-Pro-P1-Lys, showed comparable kinetic constants againstthrombin (Table II). A significant advantage of ACC substrates over AMCsubstrates is the ease of synthesizing ACC substrates over AMCsubstrates. By employing the synthesis methods described, any amino acidACC substrate can be prepared rapidly with Fmoc-based synthesisprotocols.

[0258] The major difference between the ACC and AMC libraries was theamount of enzyme and substrate required for sufficient fluorescencesignal. The substrate concentration for the ACC library was reduced to0.1 μM per substrate per well, compared to 0.25 μM for the AMC library.The enzyme concentration was also reduced. The increased fluorescencesensitivity of the ACC group will be very important for assayingproteases that are available only in limited amounts. For additionalvalidation, specific substrates that differed only in fluorogenicleaving groups, ACC or AMC, were synthesized. Steady state kineticconstants of thrombin were measured for these substrates and shown to besimilar for both the ACC and AMC containing substrates (Table II). TABLEII Substrate k_(cat) (s⁻¹) K_(m) (μM) k_(cat)/K_(m) (μM⁻¹ s⁻¹)Ac-Nle-Thr-Pro-Lys-AMC 31.0 ± 0.9  115 ± 10 0.26 ± 0.03 Ac-NleThr-Pro-Lys--ACC 33.7 ± 2.7  125 ± 13 0.28 ± 0.05 Ac-Leu-Gly-Pro-Lys-AMC2.3 ± 0.2 160 ± 25 0.015 ± 0.002 Ac-Leu-Gly-Pro-Lys--ACC 3.2 ± 0.4 195 ±30 0.018 ± 0.003

Example 7

[0259] 7.1 βII Tryptase Gene Construction

[0260] The pPIC9-Hu Try (human βI tryptase plasmid) (Niles et al.,Biotechnology and Applied Biochemistry 28 (Pt 2): 125-31 (1998)) wassubjected to site-directed mutagenesis using the GeneEditor™ in vitroSite-Directed Mutagenesis System (Promega, Madison Wis.). The mutantoligonucleotide 5′-GAGGAGCCGGTGAAGGTCTCCAGCCAC-3′ was used to introducea substitution mutation in the DNA coding for amino acid residue 113(N113K). Full-length nucleic acid sequencing of both strands confirmedthe sequence conversion to the βII tryptase isoform.

[0261] 7.2 Expression and Purification

[0262] Recombinant human βI and βII tryptases were expressed andpurified as previously described (Niles (1998)). Briefly, pPIC9-HuTry/N113K was linearized by Sac I digestion and transformed into the GS115 strain of Pichia pastoris. A tryptase expressing clone was isolatedand used for large scale expression by fermentation in buffered minimalmethanol complex media with 0.5 mg/ml heparin. Secreted mature βI andβII tryptases were purified to homogeneity using a two-column affinitychromatography procedure described previously. The enzymes weresuspended in a final storage buffer containing 2M NaCl and 10 mM MES, pH6.1 and 10% glycerol.

[0263] The proportion of catalytically active βI and βII tryptase wasquantitated by active-site titration with MUGB (Jameson et al.,Biochemical Journal 131 (1): 107-17 (1973)). Briefly, fluorescence wasmonitored, with excitation at 360 nm and emission at 450 nm, uponaddition of enzyme to MUGB. The concentration of enzyme was determinedfrom the increase in fluorescence based on a standard concentrationcurve.

[0264] The recombinant human βI and βII tryptases (1 μg) and nativehuman lung tryptase were subjected to reducing SDS/PAGE on a 4-20% TGgel (Novex). Following electrophoresis, the gel was stained by GelCode™(Pierce, Rockford, Ill.) (FIG. 4) to verify size and purity.

[0265] 7.3 Results Recombinant tryptase βI and βII were produced andsecreted in Pichia pastoris as mature enzymes. The ability to produceactive mature enzyme rather than the zymogen is important for substratespecificity studies because it obviates the need to remove thepro-peptide through the addition of an activating protease, whoseactivity may complicate subsequent specificity studies. There is asingle amino acid difference between tryptase βI and tryptase βII atposition 113, an asparagine and a lysine respectively. Replacement ofasparagine for lysine removes an N-linked glycosylation site in tryptaseβII, making it singly glycosylated. The relative degree of glycosylationcan be seen in the recombinant expression of both enzymes (FIG. 4) withtryptase βI migrating as mutiple glycosylated bands and tryptase βIImigrating as a single glycosylated band. The only difference seen inexpression and purification of the two enzymes is the final yield ofactive enzyme with tryptase βI expressing ten-fold more than tryptaseβII. The phenomenon of reduced expression upon removal of aglycosylation site has been observed with other proteases and has beenpostulated to involve decreased stability or solubility of the enzymelacking post-translational glycosylation (Harris et al., Journal ofBiological Chemistry 273(42): 27364-73 (1998)).

Example 8

[0266]8.1 Positional Scanning Synthetic Combinatorial Library Screening

[0267] Preparation and screening of the positional scanning syntheticcombinatorial library (PS-SCL) was carried out as previously described(Harris et al., Proceedings of the National Academy of Sciences 97(14):7754-7759 (2000); Backes et al., Nature Biotechnology 18(2): 187-193(2000)). The concentration of each of the 361 substrates per well in theP1-Lysine and P1-Arginine libraries was 0.25 μM. The concentration ofthe 6859 compounds per well in the P1-Diverse library was 0.013 μM.Enzyme activity of the PS-SCL was assayed in 100 mM HEPES pH 7.5, 10%glycerol and 0 or 0.1 mg/ml heparin at excitation and emissionwavelengths of 380 nm and 450 nm respectively.

[0268] 8.2 Results

[0269] To explore whether this single difference in glycosylationaffects the substrate specificity of tryptase βI and βII, threecombinatorial peptide libraries with fluorogenic leaving groups wereused. The P1-specificity was first defined using a library in which eachof the P1-amino acids in a tetrapeptide was held constant while theother three positions contain an equimolar mixture of 19 amino acids(cysteine was omitted and norleucine replaced methionine). Both tryptaseβI and βII prefer cleaving after lysine over arginine with no otheramino acids being accepted at this position (FIG. 5).

[0270] To define the extended substrate specificities of the β-tryptasesas well as to determine if extended specificity is dependent on thecontext of the P1 amino acid, tryptase βI and βII were screened againsttwo libraries that differed only in the P1 amino acid that was heldconstant, lysine and arginine. The P4 to P2 extended substratespecificities of both β-tryptases reveal that the isoforms have asimilar substrate preference that is not dependent on the P1 amino acid(FIG. 6A and FIG. 6B). Also apparent from the specificity screen is thatmany sub-optimal amino acids can be accommodated in the substratesuggesting that additional mechanisms of substrate discrimination mayalso be in place. Both tryptases show an unusual preference for prolinein the P4 position; no other serine protease has been shown to havepreference to date. The P3 position shows a preference for positivelycharged amino acids. Finally, the P2-position shows a modest preferencefor asparagine (FIG. 6A and FIG. 6B).

Example 9

[0271]9.1 Single Substrate Kinetic Analysis

[0272] Tryptase activity was monitored at 30° C. in assay buffercontaining 100 mM HEPES pH 7.5 and 10% glycerol. Substrate stocksolutions were prepared in DMSO. The final concentration of substrateranged from 0.005-2 mM. The concentration of DMSO in the assay was lessthan 5%. The tryptase concentration was 5 nM. Hydrolysis of ACCsubstrates was monitored fluorometrically with an excitation wavelengthof 380 nm and emission wavelength of 450 nm on a Fluoromax-2spectrofluorimeter (JY Horiba).

[0273] 9.2 Irreversible Inhibitor, Ac-PRNK-cmk, Kinetic Analysis

[0274] Progress curves were obtained for tryptase (1 nM) inactivation bymultiple concentrations of Ac-PRNK-cmk (50 nM to 10 μM). Activity wasmonitored at 30° C. in activity buffer with 100 μM Ac-PRNK-ACCsubstrate. The rate constant for loss of enzyme activity, k_(obs), wasdetermined from a non-linear regression of the progress curve data.k_(obs) varied linearly with inhibitor concentration. Therefore,k_(ass), the rate constant for the inactivation of enzyme withinhibitor, was determined by linear regression analysis (Bieth, J. G.Methods in Enzymology 248: 59-84 (1995)). Several P1-basic-preferringproteases were monitored for inhibition by Ac-PRNK-cmk as follows:tryptase bI (50 nM), tryptase bII (50 nM), factor Xa (50 nM), tPA (50nM), uPA (50 nM), thrombin (1 nM), and plasmin (5 nM) were incubated for5 minutes with 0 μM, 10 μM, 100 μM Ac-PRNK-cmk. After incubation,residual activity was monitored as follows: Ac-PRNK-ACC was added to afinal concentration of 5 μM to the samples containing tryptase βI andβII; Ac-GTAR-ACC (5 μM) was added to the factor Xa and tPA samples;Ac-QFAR-ACC (5 μM) was added to the uPA samples; Ac-nTPR-ACC (5 μM) wasadded to the thrombin samples; and Ac-KQWK-ACC (5 μM) was added toplasmin samples.

[0275] 9.3 Results

[0276] To quantitate tryptase βI and βII dependence on extendedsubstrate specificity, several peptide substrates were synthesized andthe kinetic constants determined for each of the enzymes. The slightpreference for lysine over arginine as seen in the P1-Diverse peptidelibrary (FIG. 4) was validated with the substrates Ac-PRNK-ACC andAc-PRNR-ACC. The Ac-PRNR-ACC substrate displays about 70-90% of theactivity of Ac-PRNK-ACC substrate; compare k_(cat)/K_(m) of(1.12±0.14)×10⁶ M⁻¹s⁻¹ to (1.23±0.15)×10⁶ M⁻¹s⁻¹ for tryptase βI and(1.31±0.19)×10⁶ M⁻¹s⁻¹ to (1.89±0.17)×10⁶ M⁻¹s⁻¹ for tryptase βII (TableIII). A minimal preference, approximately two-fold, for P2-asparagineover P2-threonine was seen for both enzymes when Ac-PRNK-ACC is comparedto Ac-PRTK-ACC, k_(cat)/K_(m) of (0.78±0.07)×10⁶ M⁻¹s⁻¹ to(1.23±0.15)×10⁶ M⁻¹s⁻¹ for tryptase βI and (1.27±0.12)×10⁶ M⁻¹s⁻¹ to(1.89±0.17)×10⁶ M⁻¹s⁻¹ for tryptase βII. A major difference is seen inthe P3-position with an approximately ten-fold preference forAc-PRNK-ACC over Ac-PANK-ACC, compare k_(cat)/K_(m) of (1.23±0.15) 10⁶M⁻¹s⁻¹ to (0.14±0.01)×10⁶ M⁻¹s⁻¹ for tryptase βI and (1.89±0.17)×10⁶M⁻¹s⁻¹ to (0.18±0.01)×10⁶ M⁻¹s⁻¹ for tryptase βI. All of these effectsare manifested in the K_(m) term, not the k_(cat) term. This indicatesthat ground state binding and recognition are important factors intryptase catalysis. These results are consistent with previous findingof Tanaka et al who showed that Z-Lys-Gly-Arg-pNA was the most optimalof the fourteen tripepidyl para-nitroanalide substrates tested (Tanakaet al., Journal of Biological Chemistry 258(22): 13552-13557 (1983)).TABLE III Substrate k_(cat) (s⁻¹) K_(m) (μM) k_(cat)/K_(m) (s⁻¹ M⁻¹) βITryptase Ac-PRNK-AAC 16.84 ± 0.27 8.9 ± 0.9 (1.89 ± 0.17) × 10⁶Ac-PANK-AAC 20.27 ± 0.48 110.5 ± 9.8  (0.18 ± 0.01) × 10⁶ Ac-PRTK-AAC18.67 ± 0.30 14.7 ± 1.4  (1.27 ± 0.12) × 10⁶ Ac-PRNR-AAC 21.75 ± 0.6716.5 ± 2.7  (1.31 ± 0.19) × 10⁶ βII Tryptase Ac-PRNK-AAC 17.84 ± 0.4014.5 ± 1.9  (1.23 ± 0.15) × 10⁶ Ac-PANK-AAC 19.06 ± 0.64 133.3 ± 15.6 (0.14 ± 0.01) × 10⁶ Ac-PRTK-AAC 18.34 ± 0.33 23.4 ± 2.3  (0.78 ± 0.07) ×10⁶ Ac-PRNR-AAC 20.94 ± 0.57 18.6 ± 2.6  (1.12 ± 0.14) × 10⁶

Example 10

[0277] 10.1 Irreversible Inhibitor, Ac-PRNK-cmk, Kinetic Analysis

[0278] Progress curves were obtained for tryptase (1 nM) inactivation bymultiple concentrations of Ac-PRNK-cmk (50 nM to 10 μM). Activity wasmonitored at 30° C. in activity buffer with 100 μM Ac-PRNK-ACCsubstrate. The rate constant for loss of enzyme activity, k_(obs), wasdetermined from a non-linear regression of the progress curve data.k_(obs) varied linearly with inhibitor concentration. Therefore,k_(ass), the rate constant for the inactivation of enzyme withinhibitor, was determined by linear regression analysis (Bieth (1995)).Several P1-basic-preferring proteases were monitored for inhibition byAc-PRNK-cmk as follows: tryptase bI (50 nM), tryptase bII (50 nM),factor Xa (50 nM), tPA (50 nM), uPA (50 nM), thrombin (1 nM), andplasmin (5 nM) were incubated for 5 minutes with 0 μM, 10 μM, 100 μMAc-PRNK-cmk. After incubation, residual activity was monitored asfollows: Ac-PRNK-ACC was added to a final concentration of 5 μM to thesamples containing tryptase βI and βII; Ac-GTAR-ACC (5 μM) was added tothe factor Xa and tPA samples; Ac-QFAR-ACC (5 μM) was added to the uPAsamples; Ac-nTPR-ACC (5 μM) was added to the thrombin samples; andAc-KQWK-ACC (5 μM) was added to plasmin samples.

[0279] 10.2 Results

[0280] To demonstrate that information obtained from the substratescreen could be translated into a potent tryptase inhibitor, theirreversible inhibitor Ac-PRNK-cmk was tested for inhibition oftryptase. The measured association rate constant, k_(ass) of 5000±200M⁻¹ sec⁻¹ for both βI and βII tryptase indicates that Ac-PRNK-cmk is apotent inhibitor of tryptase. Selectivity of the designed tryptaseinhibitor, Ac-PRNK-cmk, was demonstrated through the measurement ofinhibition of several tryptic plasma proteases, factor Xa, tPA, uPA,thrombin, and plasmin. At an inhibitor concentration of 10 μM, wheretryptase is 95% inhibited, none of the proteases tested showedinhibition (Table IV). At a 10-fold higher inhibitor concentration ofinhibitor (100 μM), where tryptase is completely inhibited, only uPA andplasmin showed inhibition, 34% and 63% inhibition respectively (TableIV). TABLE IV Percent Inhibition Percent Inhibition Enzyme 100 μMAc-PRNK-cmk 10 μM Ac-PRNK-cmk Tryptase βI 100 95 Tryptase βII 100 95Factor Xa 0 0 tPA 0 0 uPA 34 0 Thrombin 0 0 Plasmin 63 0

Example 11

[0281] 11.1 Structural Modeling of Optimized Substrate into TryptaseActive Site

[0282] The tryptase structure (PDB code 1a01) was prepared for modelingby removing inhibitor and water molecules, adding hydrogens usingSybyl6.5 (Tripos Inc. 1699 South Hanley Road, S. L., Missouri, 63144,USA.), and assigning AMBER partial atomic charges (Cornell et al.,Journal of the American Chemical Society 117(19): 5179-5197 (1995)).Because the structure was solved with a covalent inhibitor, thecatalytic Ser-195 was modeled to a geometry consistent with anon-covalent inhibitor by restoring the hydrogen bond with His-57. Thiswas accomplished with a two-step torsional minimization in Sybyl (Triposforce field, ε=1r). In the first step the position of the Ser-195hydroxyl hydrogen was minimized via torsion around the χ₂ bond, and inthe second step both the oxygen and hydrogen were minimized via torsionaround the χ₂ bond and χ₁(CCCO) bonds. The structure of the enzyme washeld rigid for the remainder of the modeling.

[0283] The capped peptide backbone of Ac-PRNK-Nme was modelled into theactive site of the tryptase structure as follows. The structure of theP1-P3 portion of ovomucoid (complexed to chymotrypsin, PDB code 1cho)was used as a template for the backbone configuration. This portion ofthe inhibitor was translated into the tryptase active site using leastsquares superposition of the protease active site residues His-57,Asp-102, Ser-195, and 214-216 onto the corresponding residues of thetryptase “A” protomer. The peptide sidechains were then truncated atC-β, hydrogens and AMBER charges were added (as above) and theconfiguration of the resultant (Ace-AAA-Nme) peptide was optimized withsuccessive minimizations in the tryptase active site. Using DOCK4.0.1(Ewing, T. J. A., Makino, S., Skillman, A. G., and Kuntz, I. D. (InPress), the atoms of the scissile amide bond were minimized first, thensuccessive rigid segments of the peptide were added (with torsionalangles taken from the ovomucoid inhibitor) alternating withminimization. The minimizations included rigid and flexible degrees offreedom and were performed using the simplex algorithm with up to 500iterations for each minimization. The DOCK energy scoring, applied toboth intermolecular and intramolecular atom pairs, includes thecoulombic and van der Waals terms from the AMBER force field (Ewing,supra; Weiner et al., Journal of Computational Chemistry 7(2): 230-252(1986)). An interatomic cutoff of 25 Å and ε=4r. The peptide side chains(PRNK) were then added, and the conformation of the P1-P3 side chainsand the P4 proline were modelled with DOCK4.0. Finally, 10 independentminimizations were carried out, and the lowest-energy configuration wasretained.

[0284] 11.2 Results

[0285] The source of the preference for basic residues at the P1position is well known for this class of proteolytic enzyme: Asp-189 ispresent in all trypsin-like serine proteases and resides at the bottomof the S1 pocket. The source of extended specificity is less apparent.The structure of tryptase is unique among serine proteases in that it isa ring-like tetramer with the four active sites in close proximitywithin the interior pore (Pereira et al., Nature 392: 306-311 (1998)).Using the program DOCK with energy scoring (Meng et al., Journal ofComputational Chemistry 13(4): 505-524 (1992)), the capped tripeptideAc-PRNK-Nme was docked into the active site of BII tryptase. The dockedmolecule had a score of −86.34 DOCK units, consisting of anelectrostatic contribution of −56.88 and a van der Waals contribution of−29.46. The unusually large electrostatic component is a result of thelarge negative charge concentrated within the pore of the tetramer.

[0286] The model of substrate binding reveals a paired binding site,with contributions from two tryptase protomers. Specifically, docking ofthe optimal peptide into the active site of tryptase predicts that theP4 and P3 side chains interact with the adjacent protomer. The P4-Proside chain interacts with the γ-carbon of Thr-96′ of the adjacentprotomer (FIG. 7). A recognition site for the P3-Arg is formed by acidicresidues from both protomers, Glu-217 from the cognate protomer andAsp-60B′ from the adjacent protomer (FIG. 7). Formation of the P4 and P3side chain interactions requires a somewhat non-canonical backboneconfiguration resulting in the loss of a backbone hydrogen bond. Bycontrast, the P2 and P1 sites make the canonical interactions seen withother members of this protease class. For example, the deep S1-pocketcontains Asp-189 from the cognate protomer that interacts with P1-Lys(FIG. 7). Another consequence of the structure is that each active sitehas an adjacent active site in close proximity leading to potentialsubstrate-substrate interactions (FIG. 7).

[0287] It is understood that the examples and embodiments describedherein are for illustrative purposes only and that various modificationsor changes in light thereof will be suggested to persons skilled in theart and are to included within the spirit and purview of thisapplication and are considered within the scope of the appended claims.All publications, patents, and patent applications cited herein arehereby incorporated by reference in their entirety for all purposes.

1 11 1 4 PRT Artificial Sequence Description of Artificial SequenceP4-P1sequence for tryptase activation of pro-urokinase plasminogen activator1 Pro Arg Phe Lys 1 2 4 PRT Artificial Sequence Description ofArtificial Sequencevasoactive intestinal peptide tryptase cleavagesequence 2 Thr Arg Leu Arg 1 3 4 PRT Artificial Sequence Description ofArtificial SequenceG-protein coupled receptor PAR-2 tryptase activationcleavage sequence 3 Ser Lys Gly Arg 1 4 4 PRT Artificial SequenceDescription of Artificial Sequencethrombin activated receptor PAR-1tryptase inactivation sequence 4 Pro Asn Asp Lys 1 5 4 PRT ArtificialSequence Description of Artificial Sequencefactor Xa P4-P1 activationcleavage sequence in prothrombin 5 Ile Glu Gly Arg 1 6 4 PRT ArtificialSequence Description of Artificial Sequencefactor Xa P4-P1 activationcleavage sequence in prothrombin 6 Ile Asp Gly Arg 1 7 4 PRT ArtificialSequence Description of Artificial Sequencefactor Xa P4-P1 activationcleavage sequence in factor VII 7 Pro Gln Gly Arg 1 8 4 PRT ArtificialSequence Description of Artificial Sequencefactor Xa P4-P1 activationcleavage sequence in autolysis loop of factor Xa 8 Glu Lys Gly Arg 1 9 4PRT Artificial Sequence Description of Artificial Sequencetetrapeptidesubstrate thrombin-susceptible sequence 9 Leu Gly Pro Lys 1 10 27 DNAArtificial Sequence Description of Artificial Sequence site-directedmutagenesis mutant oligonucleotide 10 gaggagccgg tgaaggtctc cagccac 2711 4 PRT Artificial Sequence Description of Artificial Sequencetryptaseoptimal peptide substrate 11 Pro Arg Asn Lys 1

What is claimed is:
 1. A material having a fluorogenic moiety linked toa solid support, said material having the structure:

wherein: R¹, R², R³, R⁴, R⁵ and R⁶ are members independently selectedfrom the group consisting of H, halogen, —NO₂, —CN, —C(O)_(m)R⁷,—C(O)NR⁸R⁹, —S(O)_(t)R¹⁰, —SO₂NR¹¹R¹², —OR¹³, substituted orunsubstituted alkyl, —R¹⁴-SS, and —NHR¹⁵ with the proviso that at leastone of R¹, R², R³, R⁴, R⁵ and R⁶ is —R¹⁴-SS and at least one of R¹, R²,R³, R⁴, R⁵ and R⁶ is —NHR¹⁵, wherein: R⁷, R⁸, R⁹, R¹⁰, R¹¹, R¹² and R¹³are members independently selected from the group consisting of H,substituted or unsubstituted alkyl and substituted or unsubstitutedaryl; R¹⁴ is a linking group adjoining said fluorogenic moiety and saidsolid support; R¹⁵ is a member selected from the group consisting ofamine protecting groups, —C(O)-AA and —C(O)-P: wherein: P is a peptidesequence; AA is an amino acid residue; m is a member selected from thegroup consisting of the integers 1 and 2; t is a member selected fromthe group consisting of the integers from 0 to 2; and SS is a solidsupport.
 2. The material according to claim 1, wherein said linkinggroup is a member selected from the group consisting of substituted orunsubstituted alkyl, substituted or unsubstituted heteroalkyl andsubstituted or unsubstituted aryl;
 3. The material according to claim 1,wherein P is a peptide sequence comprising the structure:—C(O)-AA¹-AA²-(AA^(i))_(J-2) wherein, AA¹-AA²-(AA^(i))_(J-2) is apeptide sequence, wherein each of AA¹ through AA^(i) is an amino acidresidue which is a member independently selected from the group ofnatural amino acid residues, unnatural amino acid residues and modifiedamino acid residues; J denotes the number of amino acid residues formingsaid peptide sequence and is a member selected from the group consistingof the numbers from 2 to 10, such that J-2 is the number of amino acidresidues in the peptide sequence exclusive of AA¹-AA²; and i denotes theposition of said amino acid residue relevant to AA¹ and when J isgreater than 2, i is a member selected from the group consisting of thenumbers from 3 to
 10. 4. The material according to claim 1, wherein R¹⁵has the structure: —C(O)-AA; andAA is an amino acid residue selectedfrom the group consisting of natural amino acids, unnatural amino acidsand modified amino acids.
 5. The material according to claim 1, havingthe structure:


6. The material according to claim 5, having the structure:

wherein, Z is a member selected from the group consisting of —O—, and—NR¹⁶—; and c is a member selected from the integers from 0 to
 6. 7. Amaterial according to claim 6, having the structure:


8. A method of assaying for the presence of an enzymatically activeprotease in a sample, said method comprising: (a) contacting said samplewith a material according to claim 3 in such a manner whereby saidfluorogenic moiety is released from said peptide sequence upon action ofsaid protease, thereby producing a fluorescent moiety; and (b) observingwhether said sample undergoes a detectable change in fluorescence, saiddetectable change being an indication of the presence of saidenzymatically active protease in said sample.
 9. The method according toclaim 8, wherein said protease is a member selected from the groupconsisting of aspartic protease, cysteine protease, metalloprotease andserine protease.
 10. The method according to claim 8, wherein saidprotease is a protease of a microorganism.
 11. The method according toclaim 10, wherein said microorganism is a member selected from the groupconsisting of bacteria, fungi, yeast, viruses, and protozoa.
 12. Themethod according to claim 8, wherein said sample is a clinical samplefrom a subject.
 13. The method according to claim 8, further comprising(c) quantifying said fluorescent moiety, thereby quantifying saidprotease.
 14. A method of assaying for the presence of a selectedmicroorganism in a sample by probing the sequence specificity of peptidecleavage by a protease of said microorganism, said method comprising:(a) contacting a sample suspected of containing said selectedmicroorganism with a material according to claim 3, wherein said peptidecomprises a sequence that is selectively cleaved by said protease ofsaid selected microorganism, thereby releasing the fluorogenic moietyfrom the peptide sequence; (b) detecting the cleavage by detectingfluorescence arising from a fluorescent moiety produced by cleavage ofsaid fluorogenic moiety from said peptide sequence, thereby confirmingsaid presence of said selected microorganism in said sample.
 15. Themethod according to claim 14, further comprising (c) quantifying saidfluorescence, thereby quantifying said protease of said microorganism.16. A fluorogenic peptide comprising a fluorogenic moiety covalentlybound to a peptide sequence, said peptide having the structure: R-Pwherein: P is a peptide sequence having the structure:—C(O)-AA¹-AA²-(AA^(i))_(J-2) wherein: each of AA¹ through AA^(i) is anamino acid residue which is a member independently selected from thegroup of natural amino acid residues, unnatural amino acid residues andmodified amino acid residues; J denotes the number of amino acidresidues forming said peptide sequence and is a member selected from thegroup consisting of the numbers from 2 to 10, such that J-2 is thenumber of amino acid residues in the peptide sequence exclusive ofAA¹-AA²; i denotes the position of said amino acid residue in sequencerelative to AA¹ and when J is greater than 2, i is a member selectedfrom the group consisting of the numbers from 3 to 10; and R is afluorogenic moiety having the structure:

wherein: R¹, R², R³, R⁴, R⁵ and R⁶ are members independently selectedfrom the group consisting of H, halogen, —NO₂, —CN,—C(O)_(m)R⁶,—C(O)NR⁷R⁸, —S(O)_(t)R⁹, —SO₂NR¹⁰R¹¹, —OR¹², substituted orunsubstituted alkyl, —NHC(O)-P, and —R²⁰—Y, with the proviso that atleast one of R¹, R², R³, R⁴, R⁵ and R⁶ is —R²⁰—Y and at least one of R¹,R², R³, R⁴, R⁵ and R⁶ is —NHC(O)-P, wherein: R⁶, R⁷, R⁸, R⁹, R¹⁰, R¹¹and R¹² are members independently selected from the group consisting ofH, substituted or unsubstituted alkyl and substituted or unsubstitutedaryl; R²⁰ is either present or absent and is a member selected from thegroup consisting of substituted or unsubstituted alkyl and substitutedor unsubstituted heteroalkyl; Y is a member selected from the groupconsisting of organic functional groups and methyl; m is a memberselected from the group consisting of the integers 1 and 2; and t is amember selected from the group consisting of the integers from 0 to 2.17. The fluorogenic peptide according to claim 16, wherein said organicfunctional group is a member selected from the group consisting of—COOR¹⁷, CONR¹⁷R²¹, —C(O)R¹⁷R²¹, —OR¹⁷, —SR¹⁷, —C(O)SR¹⁷ and —NR¹⁷R²¹wherein, R¹⁷ and R²¹ are members independently selected from H,substituted or unsubstituted alkyl and substituted or unsubstitutedaryl.
 18. A fluorogenic peptide according to claim 16, having thestructure:


19. A fluorogenic peptide according to claim 18, having the structure:

wherein: c is a member selected from the group consisting of theintegers from 0 to
 6. 20. A fluorogenic peptide according to claim 19,having the structure:


21. The fluorogenic peptide according to claim 16, wherein said peptidesequence comprises a peptide bond that is cleaved by a proteasereleasing said fluorogenic moiety from said peptide sequence, therebyproducing a fluorescent moiety and a peptide moiety.
 22. The fluorogenicpeptide according to claim 21, wherein said peptide bond is formedbetween a carboxyl of the carboxy-terminus amino acid residue and anamine group of said fluorogenic moiety.
 23. A method of assaying for thepresence of an enzymatically active protease in a sample, said methodcomprising: (a) contacting a sample suspected of containing saidprotease with a peptide according to claim 16 in such a manner wherebysaid fluorogenic moiety is released from said peptide sequence uponaction of said protease, thereby producing a fluorescent moiety; and (b)observing whether said sample undergoes a detectable change influorescence, said detectable change being an indication of the presenceof said enzymatically active protease in said sample.
 24. The methodaccording to claim 23, wherein said protease is a member selected fromthe group consisting of aspartic protease, cysteine protease,metalloprotease and serine protease.
 25. The method according to claim23, wherein said protease is a protease of a microorganism.
 26. Themethod according to claim 25, wherein said microorganism is a memberselected from the group consisting of bacteria, fungi, yeast, viruses,and protozoa.
 27. The method according to claim 23, wherein said sampleis a clinical sample from a subject.
 28. The method according to claim27, wherein said subject is a human.
 29. The method according to claim23, further comprising (c) quantifying said fluorescent moiety, therebyquantifying said protease.
 30. A method of assaying for the presence ofa selected microorganism in a sample by probing the sequence specificityof peptide cleavage by a protease of said microorganism, said methodcomprising: (a) contacting a sample suspected of containing saidselected microorganism with a material according to claim 16, whereinsaid peptide comprises a sequence that is selectively cleaved by aprotease of a selected microorganism, thereby releasing said fluorogenicmoiety from said peptide sequence; (b) detecting said cleavage bydetecting fluorescence arising from a fluorescent moiety produced bycleavage of said fluorogenic moiety from said peptide sequence, therebyconfirming said presence of said selected microorganism in said sample.31. The method according to claim 30, further comprising (c) quantifyingsaid fluorescence, thereby quantifying said protease of saidmicroorganism.
 32. A library of fluorogenic peptides comprising at leasta first peptide having a first peptide sequence covalently attached to afirst fluorogenic moiety and a second peptide having a second peptidesequence covalently attached to a second fluorogenic moiety, said firstpeptide and said second peptide having the structure: R-P wherein: foreach of said first peptide and said second peptide, P is independentlyselected from peptide sequences having the structure:—C(O)-AA¹-AA²-(AA^(i))_(J-2) wherein: each of AA¹ through AA^(i) is anamino acid residue which is a member independently selected from thegroup consisting of natural amino acid residues, unnatural amino acidresidues and modified amino acid residues; each J is independentlyselected and denotes the number of amino acid residues forming saidfirst peptide sequence and said second peptide sequence and is a memberselected from the group consisting of the numbers from 2 to 10; each iis independently selected and denotes the position of said amino acidresidue relative to AA¹ and when J is greater than 2, i is a memberselected from the group consisting of the numbers from 3 to 10; and foreach of said first peptide and said second peptide R is independentlyselected from fluorogenic moieties having the structure:

wherein: R¹, R², R³, R⁴, R⁵, and R⁶ are members independently selectedfrom the group consisting of H, halogen, —NO₂, —CN, —C(O)_(m)R⁷,—C(O)NR⁸R⁹, —S(O)_(t)R¹⁰, —SO₂NR¹¹R¹², —OR¹³, substituted orunsubstituted alkyl, —NH—C(O)-P, R²⁰—Y and —R¹⁴-SS, with the provisothat for each peptide at least one of R¹, R², R³, R⁴ and R⁵ is a memberindependently selected from —R¹⁴-SS and R²⁰—Y and at least one of R¹,R², R³, R⁴, R⁵, and R⁶ is —NH—C(O)-P, wherein: R⁷, R⁸, R⁹, R¹⁰, R¹¹, R¹²and R¹³ are members independently selected from the group consisting ofH, substituted or unsubstituted alkyl and substituted or unsubstitutedaryl; R¹⁴ is a linking group adjoining said fluorogenic moiety and thesolid support; R²⁰ is either present or absent and is a member selectedfrom the group consisting of substituted or unsubstituted alkyl andsubstituted or unsubstituted heteroalkyl; Y is a member selected fromthe group consisting of organic functional groups and methyl; m is amember selected from the group consisting of the integers from 1 to 2; tis a member selected from the group consisting of the integers from 0 to2; Y is a member selected from the group consisting of —COOR¹⁷, CONHR¹⁷,—C(O)R¹⁷, —OR¹⁷, —SR¹⁷, and NHR¹⁷, R¹⁷ is a member selected from thegroup consisting of H, substituted or unsubstituted alkyl andsubstituted or unsubstituted aryl; and SS is a solid support.
 33. Thelibrary according to claim 32, wherein said linking group is a memberselected from the group consisting of substituted or unsubstituted alkyland substituted or unsubstituted heteroalkyl
 34. The library accordingto claim 32, wherein said organic functional group is a member selectedfrom the group consisting of —COOR¹⁷, CONR¹⁷R²¹, —C(O)R¹⁷R²¹, —OR¹⁷,—SR¹⁷, —C(O)SR¹⁷, and —NR¹⁷R²¹ wherein, R¹⁷ and R² are membersindependently selected from H, substituted or unsubstituted alkyl andsubstituted or unsubstituted aryl.
 35. The library of fluorogenicpeptides according to claim 32, wherein R-P has the structure:


36. A library of fluorogenic peptides according to claim 35, wherein R-Phas the structure:

wherein, c is a member selected from the group consisting of the numbersfrom 0 to
 6. 37. A library of fluorogenic peptides according to claim36, wherein R-P has the structure:


38. The library according to claim 32, wherein said fluorogenic moietyof said first peptide and said fluorogenic moiety of said second peptideare different fluorogenic moieties.
 39. The library according to claim32, wherein said first peptide sequence and said second peptide sequenceare identical.
 40. The library according to claim 32, wherein said firstpeptide sequence and said second peptide sequence are different.
 41. Thelibrary according to claim 40, wherein an amino acid residue selectedfrom the group consisting of AA¹, AA², AA^(i) and combinations thereofof said first peptide is a different amino acid residue than an aminoacid residue at a corresponding position relative to AA¹ of said secondpeptide.
 42. The library according to claim 32, wherein AA¹ of saidfirst peptide sequence and AA¹ of said second peptide sequence areidentical amino acid residues.
 43. The library according to claim 32,wherein AA¹ of said first peptide sequence and AA¹ of said secondpeptide sequence are different amino acid residues.
 44. The libraryaccording to claim 32, wherein AA² of said first peptide sequence andAA² of said second peptide sequence are identical amino acid residues.45. The library according to claim 32, wherein AA² of said first peptidesequence and AA² of said second peptide sequence are different aminoacid residues.
 46. The library according to claim 32, wherein AA^(i) ofsaid first peptide sequence and AA^(i) of said second peptide sequenceare identical amino acid residues.
 47. The library according to claim32, wherein AA^(i) of said first peptide sequence and AA^(i) of saidsecond peptide sequence are different amino acid residues.
 48. Thelibrary according to claim 42, comprising at least six peptides havingdifferent peptide sequences, wherein AA¹ is a different amino acidresidue in each of said different peptide sequences.
 49. The libraryaccording to claim 48, comprising at least twelve peptides havingdifferent peptide sequences wherein AA¹ is a different amino acidresidue in each of said different peptide sequences.
 50. The libraryaccording to claim 49, comprising at least twenty peptides havingdifferent peptide sequences wherein AA¹ is a different amino acidresidue in each of said different peptide sequences.
 51. The libraryaccording to claim 32, wherein AA¹ is a member selected from the groupconsisting of Lys, Arg, Leu and combinations thereof.
 52. The libraryaccording to claim 32, wherein J is a member selected from the numbersfrom 4 to
 8. 53. The library of peptides according to claim 32, whereinat least one of said first peptide and said second peptide is cleavableby a protease into a fluorescent moiety and the peptide sequence. 54.The library according to claim 32, comprising at least 10 peptides,wherein each of the peptide sequences is a different peptide sequence.55. The library according to claim 54, comprising at least 100 peptides,wherein each of the peptide sequences is a different peptide sequence.56. The library according to claim 55, comprising at least 1,000peptides, wherein each of the peptide sequences is a different peptidesequence.
 57. The library according to claim 56, comprising at least10,000 peptides, wherein each of the peptide sequences is a differentpeptide sequence.
 58. The library according to claim 57, comprising atleast 100,000 peptides, wherein each of the peptide sequences is adifferent peptide sequence.
 59. The library according to claim 58comprising at least 1,000,000 peptides, wherein each of the peptidesequences is a different peptide sequence.
 60. The library according toclaim 32, wherein said first peptide is located at a first region of asubstrate and said second peptide is located at a second region of asubstrate.
 61. A method of determining a peptide sequence specificityprofile of an enzymatically active protease, said method comprising: a)contacting said protease with a library of peptides according to claim32 in such a manner whereby the fluorogenic moiety is released from thepeptide sequence, thereby forming a fluorescent moiety; b) detectingsaid fluorescent moiety; c) determining the sequence of said peptidesequence, thereby determining said peptide sequence specificity profileof said protease.
 62. The method according to claim 61, furthercomprising (d) quantifying said fluorescent moiety, thereby quantifyingsaid protease.
 63. A database comprising at least one set of peptidesequence specificity data for a protease determined using a libraryaccording to claim
 32. 64. The database according to claim 63, whereinsaid database is an electronic database.
 65. The database according toclaim 64, wherein said database is distributed on a wide area network.66. A database comprising at least one set of peptide sequencespecificity data for a protease determined using a method according toclaim
 61. 67. The database according to claim 63, wherein said databaseis an electronic database.
 68. The database according to claim 64,wherein said database is distributed on a wide area network.
 69. Themethod according to claim 61, wherein said protease is a member selectedfrom the group consisting of aspartic protease, cysteine protease, andserine protease
 70. The method according to claim 61, wherein saidprotease is a protease of a microorganism.
 71. The method according toclaim 70, wherein said microorganism is a member selected from the groupconsisting of bacteria, fungi, yeast, viruses, and protozoa.
 72. Themethod according to claim 61, further comprising (c) quantifying thefluorescent moiety, thereby quantifying said protease.
 73. A method ofpreparing a fluorogenic peptide, said method comprising: a) providing afirst conjugate comprising a fluorogenic moiety covalently bonded to asolid support, said conjugate having the structure:

 wherein, R¹, R², R^(3, R) ⁴, R⁵ and R⁶ are members independentlyselected from the group consisting of H, halogen, —NO₂, —CN,—C(O)_(m)R⁷,—C(O)NR⁸R⁹, —S(O)_(t)R¹⁰, —SO₂NR¹¹R¹², —OR¹³—NR¹⁸R¹⁹, andsubstituted or unsubstituted alkyl, with the proviso that at least oneof R¹, R², R³, R⁴, R⁵ and R⁶ is —NH₂; R⁷, R⁸, R⁹, R¹⁰, R¹¹, R¹², R¹³,R¹⁸ and R¹⁹ are members independently selected from the group consistingof H, substituted or unsubstituted alkyl and substituted orunsubstituted aryl; m is a member selected from the group consisting ofthe numbers from 1 to 2; t is a member selected from the groupconsisting of the numbers from 0 to 2; R⁵ and R⁶ are membersindependently selected from the group consisting of H and—R¹⁴—C(O)NH-SS, wherein at least one of R⁵ and R⁶ is —R¹⁴—C(O)NH-SS; R¹⁴is a member selected from the group consisting of substituted orunsubstituted alkyl and substituted or unsubstituted heteroalkyl; SS isa solid support; (b) contacting said first conjugate with a firstprotected amino acid moiety (pAA¹) and an activating agent, therebyforming a peptide bond between a carboxyl group of pAA¹ and the anilinenitrogen of said first conjugate; (c) deprotecting said pAA¹, therebyforming a second conjugate having a reactive AA¹ amine moiety; (d)contacting said second conjugate with a second protected amino acid(pAA²) and an activating agent, thereby forming a peptide bond between acarboxyl group of pAA² and said reactive AA¹ amine moiety; and (e)deprotecting said pAA², thereby forming a third conjugate having areactive AA² amine moiety.
 74. The method according to claim 73, furthercomprising: (f) contacting said third conjugate with a third protectedamino acid (pAA³) and an activating agent, thereby forming a peptidebond between a carboxyl group of pAA³ and said reactive AA² aminemoiety; and (e) deprotecting said pAA³, thereby forming a fourthconjugate having a reactive AA³ amine moiety.
 75. The method accordingto claim 73, further comprising between steps (b) and (c) cappinganiline amine groups that have not reacted with pAA¹.
 76. The methodaccording to claim 75, wherein said capping utilizes a mixturecomprising an active ester of a carboxylic acid.
 77. The methodaccording to claim 78, wherein said active ester is the nitrotriazoleester of acetic acid.
 78. The method according to claim 74, wherein amember selected from the group consisting of pAA¹, pAA², pAA³ andcombinations thereof comprises a mixture of protected amino acidsdiffering in the identity of the amino acid portion of the protectedamino acids.
 79. The method according to claim 78, wherein said mixturecomprises at least 2 unique amino acids.
 80. The method according toclaim 79, wherein said mixture comprises at least 6 unique amino acids.81. The method according to claim 80, wherein said mixture comprises atleast 12 unique amino acids.
 82. The method according to claim 81,wherein said mixture comprises at least 20 unique amino acids.
 83. Themethod according to claim 78, wherein said mixture is an isokineticmixture.